CN114645023B

CN114645023B - System and method for reprogramming peripheral blood mononuclear cells to induced pluripotent stem cells

Info

Publication number: CN114645023B
Application number: CN202210541037.XA
Authority: CN
Inventors: 高歌; 周安宇
Original assignee: Ixcell Biotechnology Co ltd
Current assignee: Ixcell Biotechnology Co ltd
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2022-09-20
Anticipated expiration: 2042-05-18
Also published as: CN114645023A

Abstract

The invention discloses a reprogramming system and a culture method for reprogramming Peripheral Blood Mononuclear Cells (PBMCs) into human induced pluripotent stem cells. The reprogramming system comprises a reprogramming induction culture solution and one or more vectors capable of expressing human transcription factor OCT4, a p53 gene expression inhibitor and a Cohesin gene expression inhibitor; wherein the reprogramming induction culture fluid comprises IX-PSC basal medium, GSK-3 inhibitor, deacetylase inhibitor, MEK inhibitor, ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid. By using the system and the culture method, the reprogramming efficiency of the cells can be improved, the preparation period of the human induced pluripotent stem cells is shortened, and the preparation cost of the human induced pluripotent stem cells is saved.

Description

System and method for reprogramming peripheral blood mononuclear cells to induced pluripotent stem cells

Technical Field

The invention belongs to the technical field of induced pluripotent stem cell culture, and particularly relates to a system and a method for reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

Background

The embryonic stem cell is a pluripotent stem cell, has the potential of differentiating into various types of cells of various tissues and organs, can be applied to organ tissue transplantation and cell therapy in clinic, and can also be used for providing a cell model for researching diseases. However, since the inner cell mass of the blastocyst in the early stage of embryonic development is derived, the operation of the egg cells is inevitable during the establishment of the blastocyst, so that a plurality of ethical problems are involved and are always controversial.

The Yamanaka laboratory of Kyoto university in Japan in 2006 utilizes four transcription factors, Oct3/4, Sox2, Klf4 and c-Myc 4 to reprogram mouse fibroblasts into induced multifunctional stem cells, and marks the emergence of a novel embryonic stem cell. Induced Pluripotent Stem Cells (iPSCs) can be prepared from almost all adult tissue cells, so that the preparation of autologous pluripotent stem cells of patients becomes possible, and the limitation of clinical application of embryonic stem cells can be effectively avoided. Therefore, the iPS cell has wide application prospect in the aspects of new drug screening, in-vitro disease model establishment, cell replacement therapy and regenerative medicine.

At present, a plurality of iPSC reprogramming methods are developed internationally, but all the methods have certain limitations. If Sendai virus or lentivirus is used to carry different exogenous transcription factors to express in cells so as to start cell reprogramming, the method has the disadvantages that the immediate insertion of genome may bring potential risks and the virus induction experiment cost is high. The reprogramming component is deleted by transiently expressing Cre recombinase after induction is finished, the system can delete exogenous genes after reprogramming is finished, activation of the reprogramming factor can be avoided, and therefore the risk of tumor generation is reduced.

In addition, in order to provide sufficient nutrition for cell growth, a large amount of animal-derived components are added into a culture medium used in the existing reprogramming process of induced pluripotent stem cells, so that the clinical application of the iPSC is limited.

In a word, the existing technical system for inducing human body cells to be iPS cells generally faces the problems of low efficiency, long induction time, high preparation cost and the like. In order to widely apply human iPS cells in regenerative medicine and disease occurrence mechanism research, an efficient induction strategy, namely a reprogramming culture medium and a reprogramming method capable of shortening induction time of the iPS cells, needs to be explored.

Disclosure of Invention

The invention provides a reprogramming system and a reprogramming method for reprogramming Peripheral Blood Mononuclear Cells (PBMCs) into human induced pluripotent stem cells, aiming at the technical problems of low reprogramming efficiency and long induction time of the existing iPS cells.

One aspect of the present invention provides a reprogramming system for reprogramming Peripheral Blood Mononuclear Cells (PBMCs) into human induced pluripotent stem cells, characterized in that the reprogramming system comprises a reprogramming induction culture solution and one or more vectors capable of expressing human transcription factor OCT4, a p53 gene expression inhibitor, and a Cohesin gene expression inhibitor; wherein the reprogramming induction culture fluid comprises IX-PSC basal medium, GSK-3 inhibitor, deacetylase inhibitor, MEK inhibitor, ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid; wherein the IX-PSC basic culture medium is DMEM/F12 as basic culture medium, and is added with ascorbic acid, FGF2, TGF beta 1, neuregulin 1, IGF-1, superoxide dismutase, catalase, human serum albumin, transferrin and sodium selenite.

In some embodiments, the inhibitor of p53 gene expression and/or inhibitor of Cohesin gene expression is an inhibitory RNA molecule; preferably, the inhibitory RNA molecule is an antisense RNA, miRNA, siRNA or shRNA; more preferably, the inhibitory RNA molecule is a shRNA.

In some embodiments, the one or more vectors are non-genomically integrated vectors; more preferably, the one or more vectors are plasmids.

In some embodiments, the GSK-3 inhibitor is CHIR99021, the deacetylase inhibitor is VPA, the MEK inhibitor is PD0325901, and/or the ROCK inhibitor is Thiazovivin.

In some embodiments, in the reprogramming induction culture fluid, the human serum albumin concentration is 1-10mg/mL, the GSK-3 inhibitor concentration is 0.5-3 μ Μ, the deacetylase inhibitor concentration is 0.3-3 μ Μ, the MEK inhibitor concentration is 10-100nM, the ROCK inhibitor concentration is 0.5-5 μ Μ, and/or the 4-hydroxyethylpiperazine ethanesulfonic acid concentration is 1-10 mM;

preferably, in the reprogramming induction culture fluid, the human serum albumin concentration is 5mg/mL, the GSK-3 inhibitor concentration is 2 μ M, the deacetylase inhibitor concentration is 2 μ M, the MEK inhibitor concentration is 50nM, the ROCK inhibitor concentration is 2.5 μ M, and/or the 4-hydroxyethylpiperazine ethanesulfonic acid concentration is 5 mM.

Another aspect of the invention provides a method of reprogramming Peripheral Blood Mononuclear Cells (PBMCs) to induced pluripotent stem cells, the method comprising:

i) transfecting peripheral blood mononuclear cells with one or more vectors, wherein the one or more vectors can express human transcription factors OCT4, p53 gene expression inhibitors and Cohesin gene expression inhibitors, and obtaining peripheral blood mononuclear cells expressing the human transcription factors OCT4, p53 gene expression inhibitors and the Cohesin gene expression inhibitors;

ii) culturing the peripheral blood mononuclear cells in a reprogramming induction culture solution to obtain induced pluripotent stem cells;

wherein the reprogramming induction culture fluid comprises IX-PSC basal medium, GSK-3 inhibitor, deacetylase inhibitor, MEK inhibitor, ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid; wherein the IX-PSC basal medium is prepared by taking DMEM/F12 as a basal medium and adding ascorbic acid, FGF2, TGF beta 1, neuregulin 1, IGF-1, superoxide dismutase, catalase, human serum albumin, transferrin and sodium selenite.

In some embodiments, the total concentration of the one or more carriers is 1-3 μ g carrier/1.0 × 10 ⁵ Blood cells, preferably 2. mu.g of carrier/1.0X 10 ⁵ Blood cells.

In some embodiments, the GSK-3 inhibitor is CHIR99021, the deacetylase inhibitor is VPA, the MEK inhibitor is PD0325901 and/or the ROCK inhibitor is Thiazovivin.

In some embodiments, in the reprogramming induction culture fluid, the human serum albumin concentration is 1-10mg/mL, the GSK-3 inhibitor concentration is 0.5-3 μ Μ/mL, the deacetylase inhibitor concentration is 0.3-3 μ Μ/mL, the MEK inhibitor concentration is 10-100nM, the ROCK inhibitor concentration is 0.5-5 μ Μ, and/or the 4-hydroxyethylpiperazineethanesulfonic acid concentration is 1-10 mM;

In some embodiments, the method comprises the steps of:

step 1): peripheral Blood Mononuclear Cells (PBMC) are treated at 35-39 ℃ with 3-7% CO ₂ And 0.5-2X 10 ⁶ Culturing the cells/well in a well plate for 1-3 days;

step 2): according to 1-3. mu.g/1.0X 10 of each carrier ⁵ Blood cells the one or more vectors are transfected into the peripheral blood mononuclear cells obtained in step 1), and the cells are inoculated onto a tissue culture plate and cultured in StemPro-34 medium at 35-39 ℃ with 3-7% CO ₂ And 3 to 7% of O ₂ Culturing for 1-3 days;

and step 3): day 2-3 after transfection, fresh StemPro-34 medium was used instead; then replacing the reprogramming induction culture solution every 1-3 days, and carrying out 3-7% CO treatment at the temperature of 35-39 DEG C ₂ And 3 to 7% of O ₂ Until the induced pluripotent stem cell clone appears;

preferably, the method comprises the steps of:

step 1): peripheral Blood Mononuclear Cells (PBMC) were incubated at 37 ℃ with 5% CO ₂ And 1X 10 ⁶ Culturing the cells/well in a well plate for 2 days;

step 2): according to a total concentration of 2. mu.g carrier/1.0X 10 ⁵ Blood cells the vector or vectors are transfected into peripheral blood mononuclear cells, which are plated onto tissue culture plates using StemPro-34 medium at 37 ℃ with 5% CO ₂ And 5% of O ₂ Culturing for 2 days under the conditions of (1);

step 3): day 2 post-transfection, fresh StemPro-chambers-34 were replaced; thereafter, the reprogramming induction medium was replaced every 2 days at 37 ℃ with 5% CO ₂ And 5% of O ₂ Until the induced pluripotent stem cell clone appears;

optionally, the method further comprises: step 4): replacement of the reprogramming induction culture after the occurrence of induced pluripotent stem cell cloningNutrient solution with 3-7% CO at 35-39 deg.C ₂ The culture was continued with replacement of the stem cell medium every 1 to 2 days during the culture.

Yet another aspect of the present invention provides a kit comprising any one of the reprogramming systems described above.

A further aspect of the invention provides the use of any one of the reprogramming systems described above or any one of the kits described above for inducing reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) into human induced pluripotent stem cells.

The invention has the positive effects that: according to the invention, a GSK-3 inhibitor, a deacetylase inhibitor, a MEK inhibitor and a ROCK inhibitor are added into a reprogramming induction culture medium during cell reprogramming, and a human transcription factor OCT4, a p53 gene expression inhibitor and a Cohesin gene expression inhibitor are used for promoting cell reprogramming, so that in the process of reprogramming PBMC (peripheral component interconnect) to iPS (induced pluripotent stem cells), all components act synergistically, the cell growth and proliferation are maintained, and the cell induced reprogramming efficiency is improved. In a preferred embodiment of the invention, non-genomically integrated vectors are used, and the risk of random gene insertion encountered in conventional viral transfection is also avoided.

Drawings

FIG. 1 is a microphotograph of Peripheral Blood Mononuclear Cells (PBMC) obtained by separation;

FIG. 2 shows the number of clones obtained by different reprogramming methods;

FIG. 3 is a photograph showing the EB differentiation assay of the obtained cell line;

FIG. 4 is a photograph showing the identification of the pluripotent immunofluorescence of the obtained cell line;

FIG. 5 is a photograph showing the flow identification of the obtained cell line;

FIG. 6 is a photograph showing the karyotype identification of the obtained cell line.

Detailed description of the preferred embodiments

The present invention provides a reprogramming system for inducing reprogramming of PBMCs into induced pluripotent stem cells. The reprogramming system is a combination of multiple components, wherein each component can synergistically induce the reprogramming of the cells, so that the reprogramming efficiency of the cells is improved, and the time for inducing the reprogramming is shortened. The IX-PSC basal medium containing human serum albumin can provide abundant nutrition and necessary cytokines for cell growth and proliferation. The GSK-3 inhibitor CHIR99021 can inhibit the activity of GSK-3 alpha and GSK-3 beta in a mode of competing ATP binding sites, so that the apoptosis is inhibited, the survival rate of cells in the reprogramming process is improved, a WNT signal channel is enhanced, and the reprogramming efficiency of somatic cells into iPSCs is enhanced. The deacetylase inhibitor VPA can promote histone acetylation, so that chromatin adopts a loose structure, thereby promoting the combination of transcription factors and improving the reprogramming efficiency. The MEK inhibitor PD0325901 can achieve the effect of inhibiting MEK by changing conformation of ATP binding sites through binding with MEK, and remarkably improves survival, proliferation and reprogramming efficiency of iPSC by inhibiting MEK-ERK which regulates signal pathways related to cell proliferation and apoptosis. The ROCK inhibitor Thiazovivin can enhance the survival of embryonic stem cells, improve the generation efficiency of iPSC and shorten the reprogramming induction time of iPSC cells. 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) can maintain a stable pH value during cell culture, thereby promoting the growth and proliferation of cells. The p53 gene expression inhibitor and the Cohesin gene expression inhibitor can promote cell survival in the reprogramming process, and simultaneously can remarkably change the three-dimensional structure of chromatin to make the chromatin loose and open, thereby enhancing the action frequency of OCT4 and added induced small molecules (such as VPA, CHIR99021 and PD 0325901) and remarkably accelerating the reprogramming process.

The term "Peripheral Blood Mononuclear Cells (PBMC)" as used herein means mononuclear cells isolated from peripheral blood, and includes mainly lymphocytes, and also includes a small number of monocytes, plasma cells, hematopoietic stem cells and other progenitor cells. PBMCs for use in the present invention may be PBMCs isolated from blood or cryopreserved. PBMCs isolated from blood can generally be isolated from a cryopreserved or freshly collected blood sample by Ficoll-Paque density gradient centrifugation, e.g., in some embodiments, a cryopreserved or freshly collected blood sample can be isolated from a Ficoll-Paque density gradient centrifugationPeripheral Blood Mononuclear Cells (PBMC) with 3-7% CO at 35-39 deg.C preferably at 37 deg.C ₂ Preferably 5% CO ₂ 、0.5-2×10 ⁶ The cell density is preferably 1X 10 ⁶ The cells/well are inoculated into an ultra-low adsorption 24-well plate for culture for 1-3 days, preferably 2 days, and the obtained PBMCs can be used for transfection of the vector. In other embodiments, cryopreserved PBMCs are used, e.g., cryopreserved and then reconstituted for transfection of vectors, e.g., in some embodiments, cryopreserved PBMCs are taken and rapidly thawed in a 37 ℃ water bath. Cells were gently pipetted 1-2 times using a 1mL pipette and transferred to a 15mL centrifuge tube. Rinsing the frozen tube with 1mL of room temperature preheated StemPro-34 basal medium for 1 time, transferring the rinsed cell suspension into the centrifuge tube, and adding 9mL of StemPro-34 basal medium into the centrifuge tube drop by drop; centrifuge at 200g for 3 min. Discarding supernatant, suspending cells by StemPro-34 complete culture medium, inoculating into ultra-low adsorption 24-well plate, and selecting 3-7% CO at 35-39 deg.C preferably 37 deg.C ₂ Preferably 5% CO ₂ Culturing in an incubator for 1-3 days, preferably 2 days. In some embodiments, the PBMCs may be human PBMCs.

"reprogramming" as used herein refers to the process of dedifferentiating a somatic cell into a pluripotent stem cell.

The reprogramming induction culture solution is prepared by adding a GSK-3 inhibitor, a deacetylase inhibitor, a MEK inhibitor, a ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid into an IX-PSC basic culture medium. The IX-PSC basic culture medium is obtained by taking DMEM/F12 as a basic culture medium and adding ascorbic acid, FGF2, TGF beta 1, neuregulin 1, IGF-1, superoxide dismutase, catalase, human serum albumin, transferrin and sodium selenite. In a preferred embodiment, the IX-PSC basal medium may be supplemented with 10-30. mu.g/mL ascorbic acid, 30-50 ng/mL FGF2, 0.5-2 ng/mL TGF β 1, 3-9 ng/mL neuregulin 1, 10-20 ng/mL IGF-1, 2-3. mu.g/mL superoxide dismutase, 2-3. mu.g/mL catalase, 1-10mg/mL human serum albumin, 10-30. mu.g/mL transferrin, and 10-30. mu.g/mL sodium selenite, based on DMEM/F12. In a more preferred embodiment, the IX-PSC basal medium may be DMEM/F12 supplemented with 20. mu.g/mL ascorbic acid, 40ng/mL FGF2, 1ng/mL TGF β 1, 6ng/mL neuregulin 1, 15ng/mL IGF-1, 2.5. mu.g/mL superoxide dismutase, 2.5. mu.g/mL catalase, 5mg/mL human serum albumin, 20. mu.g/mL transferrin, and 20. mu.g/mL sodium selenite.

"DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)" as described herein is a 1:1 Mixture of DMEM and Ham's F-12, a widely used basal Medium that can be used to support the growth of a variety of mammalian cells, and is commercially available from a variety of suppliers (e.g., Thermo Fisher Scientific).

The FGF2 of the present invention is Fibroblast Growth Factor (Fibroblast Growth Factor) 2.

"TGF β 1" according to the present invention refers to Transforming Growth Factor β 1 (Transforming Growth Factor β 1).

The "Neuregulin 1" of the present invention may also be referred to as "NGR 1 (Neuredulin-1").

The term "IGF-1" as used herein refers to Insulin-like growth factor-1 (Insulin-like growth factor-1).

The reprogramming induction medium of the present invention contains 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) at a concentration of 1 to 10mM, preferably 5 mM.

The term "ROCK inhibitor" as used herein refers to an inhibitor of Rho-associated protein kinase (ROCK), including but not limited to Thiazovivin, Y-27632, and KD-025 (also referred to as SLx-2119). One or more ROCK inhibitors may be included in the culture broth of the invention. The ROCK inhibitor may be contained in the culture solution of the present invention in an amount of 0.5 to 5. mu.M, preferably 2 to 2.5. mu.M, more preferably 2.5. mu.M.

The "MEK inhibitor" of the present invention refers to an inhibitor of mitogen-activated protein kinase (MAPK/ERK kinase), including but not limited to PD 0325901. One or more MEK inhibitors may be included in the culture fluids of the present invention. The MEK inhibitor may be present in the culture medium of the invention in an amount of 10-100nM, preferably 25-50nM, more preferably 50 nM.

The "deacetylase inhibitor" according to the present invention may also be referred to as Histone deacetylase inhibitor (HDACI), which is an agent capable of inhibiting Histone Deacetylase (HDAC) activity, and includes, but is not limited to, VPA (Valproic acid), NaBu (sodium butyrate), TSA (Trichostatin a), and SAHA (suberoylanilide hydroxamic acid). One or more deacetylase inhibitors can be included in the culture broth of the invention. The content of the deacetylase inhibitor in the culture solution of the present invention may be 0.3 to 3. mu.M, preferably 1 to 2. mu.M, more preferably 1.5 to 2. mu.M, and still more preferably 2. mu.M.

The "GSK-3 inhibitor" refers to an inhibitor of Glycogen synthase kinase-3 (Glycogen synthase kinase-3), and includes but is not limited to CHIR99021, SB 216763, TWS119, LY2090314, Tideglusib and a small molecule compound BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3-oxide). One or more GSK-3 inhibitors may be included in the culture broth of the invention. The ROCK inhibitors, AHR inhibitors, deacetylase inhibitors, and GSK-3 inhibitors of the present invention are all commercially available from a variety of suppliers. The content of the GSK-3 inhibitor in the culture solution of the present invention may be 0.5 to 3. mu.M, preferably 1 to 2. mu.M, and more preferably 2. mu.M.

The reprogramming induction culture solution can be further added with trace elements B and C, and the trace elements B and C can participate in the synthesis of protein and the metabolism of nucleic acid and can also promote the survival of cells.

The trace element B is a composition of multiple trace elements, and preferably comprises ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride and hydrochloric acid. The Trace element B may be purchased from a supplier, such as Trace Elements B from Corning, cat # 25-022.

The trace element C is a composition of multiple trace elements, and preferably comprises aluminum chloride, barium acetate, cadmium chloride, chromium chloride, cobalt dichloride, germanium dioxide, potassium bromide, potassium iodide, rubidium chloride, silver nitrate, sodium fluoride and zirconyl chloride. The Trace element C may be purchased from a supplier, such as Trace Elements C from Corning, cat # 25-023.

The dilution concentrations of the trace elements B and C are 300-1000, preferably 500. Wherein the dilution concentration refers to the dilution factor when added to the medium, e.g., "300-" 1000 × "means 300-" 1000 × "when added to the medium, and similarly" 500 × "means 500 ×" when added to the medium.

The reprogramming system and method of the present invention include delivering human transcription factor OCT4, a p53 gene expression inhibitor, and a Cohesin gene expression inhibitor into a cell through a vector. Therefore, the reprogramming system of the present invention comprises one or more vectors for expressing human transcription factor OCT4, a p53 gene expression inhibitor, and a Cohesin gene expression inhibitor. When the vector or vectors are transferred into cells (e.g., PBMCs), the human transcription factor OCT4, the inhibitor of p53 gene expression, and the inhibitor of Cohesin gene expression can be expressed in PBMCs.

The term "OCT 4" refers to an octamer-binding transcription factor 4 (OCT-binding transcription factor 4), also known as OCT3 or OCT3/4, is a protein that contains a POU domain. See A K Ryan and M G Rosenfeld, Genes Dev. 1997, 11, 1207-. In some embodiments, the OCT3/4 is human OCT3/4 (hOCT 3/4). In some embodiments, the coding sequence of hhct 3/4 is:

ATGGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGTGATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGCTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCTGAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGGCGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAGACCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGGCCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGAAGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAACTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACACAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAAACGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCAGGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAACCAGTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCGAAACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGGATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCAGCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGGACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGGAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCTTTCCCCCTGTCTCCGTCACCACTCTGGGCTCTCCCATGCATTCAAACTGATGATGA（SEQ ID NO:1）

the term "p 53 gene" is a tumor suppressor gene, encoding the p53 protein, p53 protein and p53 gene are well known to those skilled in the art. In some embodiments, the sequence of the shRNA that inhibits expression of the p53 gene is CCGGGTCCAGATGAAGCTCCCAGAACTCGAGTTCTGGGAGCTTCATCTGGACTTTTT (SEQ ID NO: 2)

The term "Cohesin gene" refers to the gene encoding the chromosomal adhesion protein (Cohesin) that is commonly used to link the circular structural backbone of chromosomes, and Cohesin proteins and their encoding genes are also well known to those skilled in the art. In some embodiments, the mRNA sequence of Cohesin can be found in GenBank Accession number NC-000008. In some embodiments, the sequence of shRNA that inhibits Cohesin gene expression CACCGCCTTCATGTTGTTTGCATTCCGAAGAATGCAAACAACATGAAGGC (SEQ ID NO: 3)

In the present invention, the "gene expression inhibitor" refers to an inhibitory molecule capable of inhibiting the expression of a target gene, and this molecule can inhibit the production of mRNA or degrade mRNA by a mechanism such as, for example, inhibiting the production of mRNA or degrading mRNA, so that mRNA or protein encoded by the gene cannot be expressed by the gene, or so that the amount of expressed mRNA or protein is reduced, for example, by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, relative to the case where the inhibitor is not used. The "gene expression inhibitor" may be a molecule that inhibits the expression of a target gene by antisense inhibition or RNAi, for example, an inhibitory RNA molecule, and examples thereof include antisense RNA, siRNA, shRNA, miRNA, and the like. It will be appreciated that the inhibitor of gene expression may also be in other forms, such as a dominant negative mutant of the gene of interest, and the like. In the present invention, the gene expression inhibitor may have a specific inhibitory effect on a target gene. By "specifically inhibits" is generally meant that the inhibitor inhibits the target gene without inhibiting or substantially inhibiting other genes.

As known to those skilled in the art, the term "RNAi" refers to RNA interference, a biological process mediated by a nucleic acid molecule that inhibits or down-regulates gene expression in a cell.

As known to those skilled in the art, the term "shRNA" refers to a double-stranded structure formed from single strands of RNA that are self-complementary. shRNA constructs containing a nucleotide sequence identical to a portion of the coding or non-coding sequence of a target gene can be used to inhibit expression of the target gene. When the shRNA is expressed in a cell, it is processed through a series of steps into small interfering rna (sirna) for directing gene silencing. The shRNA can have a length of about 50-100 bp. The duplex forming portion of the shRNA may be at least 20, 21 or 22 nucleotides in length.

As known to those skilled in the art, the term "siRNA" refers to small inhibitory RNA duplexes (typically between 17-30 base pairs, but also longer base pairs such as 31-50 bp) that induce the RNA interference (RNAi) pathway.

As known to those skilled in the art, the term "antisense RNA" refers to an RNA transcript complementary to all or part of a target mRNA that can block the expression of a target gene by interfering with the processing, transport and/or translation of the mRNA. The antisense RNA can be complementary to any portion of the target mRNA, including 5 'non-coding sequences, 3' non-coding sequences, introns, and coding sequences.

As known to those skilled in the art, the term "miRNA" refers to a single-stranded RNA molecule (or synthetic derivative thereof) that is capable of binding to a target gene (mRNA or DNA) and regulating the expression of that gene.

The term "vector" in the present invention generally refers to an expression vector, which can be used to express a target gene. In a preferred embodiment, the vector is a non-integrating vector, i.e., does not integrate into the genome of the cell into which it is transfected. Non-integrative vectors are well known to those skilled in the art, such as, but not limited to, plasmids, non-integrative recombinant viral vectors (e.g., non-integrative adenoviral vectors or adeno-associated viral vectors, etc.). Expression vectors typically include a gene of interest to be expressed and regulatory sequences, such as promoters, enhancers, post-transcriptional regulatory sequences, and the like, operably linked to the gene of interest.

In the invention, the human transcription factor OCT4, the p53 gene expression inhibitor and the Cohesin gene expression inhibitor can be expressed in one vector, two vectors or three vectors respectively. For example, any two of the human transcription factor OCT4, the p53 gene expression inhibitor, and the Cohesin gene expression inhibitor (e.g., human transcription factor OCT4 and p53 gene expression inhibitor) may be expressed using one vector, and the other vector may be expressed using the other vector. For another example, three vectors each expressing one of human transcription factor OCT4, a p53 gene expression inhibitor, and a Cohesin gene expression inhibitor may be used.

The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA molecule, into a cell, preferably a eukaryotic cell, for example into a mammalian cell, such as into a PBMC cell. "transfection" can be carried out by any method known to those skilled in the art for transferring DNA (e.g., plasmid vector) into cells, for example, by a competent cell method, an artificial liposome method, a microinjection method, a particle gun method, or an electroporation method (also referred to as electroporation, electrotransformation, or electrotransfection in the present invention), and the like.

After the one or more vectors are used for transfecting the peripheral blood mononuclear cells, the transfected mononuclear cells can express human transcription factor OCT4, a p53 gene expression inhibitor and a Cohesin gene expression inhibitor, wherein the expression of the p53 gene expression inhibitor and the Cohesin gene expression inhibitor in the peripheral blood mononuclear cells can inhibit the expression of a p53 gene and a Cohesin gene in the peripheral blood mononuclear cells.

The peripheral blood mononuclear cells may be cultured in advance for an appropriate period of time to activate the cells, for example, for 1 to 3 days, preferably 2 days, before transfection. The pre-culture conditions areAs is well known to those skilled in the art, for example, 3-7% CO at 35-39 deg.C, preferably 37 deg.C ₂ Preferably 5% CO ₂ 、0.5-2×10 ⁶ The cells/well are preferably 1X 10 ⁶ The density of cells/well is cultured. The pre-incubation may be performed using any suitable vessel, for example, the incubation may be performed in an ultra-low adsorption well plate. The preculture may be carried out using any suitable medium, for example, StemPro-34 medium.

When the vector or vectors are used to transfect peripheral blood mononuclear cells, the amount of the vector to be used may be determined in accordance with the actual situation, and may be, for example, 1 to 3. mu.g of the total amount of the vector per 1.0X 10 ⁵ Peripheral blood mononuclear cells, preferably 2. mu.g of total vector/1.0X 10 ⁵ Individual cells of peripheral blood. When more than one carrier is used, the ratio between the carriers may be determined as the case may be, and may be, for example, an equal mass ratio, i.e., a mass ratio of 1: 1.

After transfection, the peripheral blood mononuclear cells transfected by the vector may be cultured directly using the reprogramming induction culture solution of the present invention, or may be cultured in another suitable medium (e.g., StemPro-34 medium) for a certain period of time, and then replaced with the reprogramming induction culture solution of the present invention. For example, in some embodiments, transfected peripheral blood mononuclear cells may be cultured for 1-3 days, preferably 2 days, using other suitable media, and then replaced with the reprogramming induction medium of the present invention. The culture conditions may be those commonly used in the art for reprogramming PBMC, and may be, for example, 35 to 39 ℃ preferably 37 ℃ and 3 to 7% CO ₂ Preferably 5% CO ₂ 、3~7% O ₂ Preferably 5% O ₂ The culturing may be carried out using any suitable vessel, e.g.on a laminin-coated plate, e.g.1. mu.g/cm coated ² The method of Lamnin521 on a plate.

The conditions for culturing peripheral blood mononuclear cells in the reprogramming induction culture solution of the present invention may be conditions generally used in the art for reprogramming PBMC, and may be, for example, 35 to 39 ℃, preferably 37 ℃ and 3 to 7% CO ₂ Preferably 5% CO ₂ 、3~7% O ₂ Preferably 5% O ₂ Under the conditions of (1). During the culture, the fresh reprogramming induction culture solution may be replaced at an appropriate time according to circumstances, and for example, the fresh reprogramming induction culture solution may be replaced every 1 to 3 days, preferably every 2 days.

The skilled person knows how to judge the occurrence of Induced Pluripotent Stem Cell (iPSC) clones, for example, morphological judgment can be performed according to unique characteristics of human embryonic stem cells (ES cells), for example, cell clones with flat, homogeneous and distinct boundary contour, i.e., induced pluripotent stem cell clones, occur.

After the induced pluripotent stem cell clone has appeared, the culture may be continued in the reprogramming inducer of the present invention in order to promote the growth thereof, and the culture conditions are well known to those skilled in the art, and may be, for example, 35 to 39 ℃ preferably 37 ℃ with 3 to 7% CO ₂ Preferably 5% CO ₂ For example, the stem cell culture medium may be replaced every 1 to 2 days, preferably 1 day, during the culture.

After the induced pluripotent stem cell clones have grown, individual clones may be picked and plated for further culture, and optionally subcultured to purify the induced pluripotent stem cells and expand their numbers for subsequent manipulation (e.g., detection or further use). The conditions for culturing and subculturing of induced pluripotent stem cells are well known to those skilled in the art, and for example, the induced pluripotent stem cell clones may be picked and seeded into a new matrigel-coated plate at 35-39 deg.C, preferably 37 deg.C, with 3-7% CO ₂ Preferably 5% CO ₂ The subculture is continued under the condition that the medium can be replaced every 1-2 days, preferably every day, and every 3-5 days, preferably every 3 days, wherein the medium can be the reprogramming induction culture solution or other stem cell culture medium of the present invention.

Examples

The technical solution of the present invention will be described in further detail below by way of examples with reference to the accompanying drawings, but the present invention is not limited to the following examples.

Example 1 cloning by different reprogramming methods

Experimental groups 1-3 PBMC cells were transfected (i.e., electroporated) using reprogramming plasmid electroporation, comprising the steps of:

step 1): the frozen PBMC is taken and rapidly thawed in a water bath at 37 ℃. Cells were gently pipetted 1-2 times using a 1mL pipette and transferred to a 15mL centrifuge tube. Rinsing the frozen tube with 1mL of room temperature preheated StemPro-34 basal medium for 1 time, transferring the rinsed cell suspension into the centrifuge tube, and adding 9mL of StemPro-34 basal medium into the centrifuge tube drop by drop; centrifuge at 200g for 3 min. The supernatant was discarded, cells were resuspended in StemPro ™ full-medium resuspension medium, counted and plated onto ultra-low adsorption 24-well plates. PBMC at 37 ℃ with 5% CO ₂ Culturing in an incubator for 2 days.

Step 2): PBMC were electrotransfected, approximately 1 hour prior to adding 2mL StemPro ™ complete medium to 1 well of a 6-well plate, and the plate was equilibrated in a 37 ℃ incubator. Collecting PBMC in a 24-well plate, rinsing with 1mL of StemPro-34 basal medium, centrifuging at 200g for 3 min; the supernatant was discarded, washed with DPBS resuspension, and counted. Centrifuging at 200g for 3 min; during centrifugation, 5mL of electrotransfer buffer E was added to the electrotransfer cup. Setting the electric transfer parameters as follows: 1650V, 10ms, 3 pulses; after centrifugation, the DPBS was aspirated and the cells were resuspended in a ratio of two plasmids equal (mass ratio) and in a total amount of 2. mu.g plasmid/1.0X 10 ⁵ Adding reprogramming plasmids into blood cells, uniformly mixing, completing electrotransfer according to electrotransfer parameters, and recording as day 0; the cells after electroporation were inoculated to 1. mu.g/cm ² The Lamnin 521-coated 6-well tissue culture plate containing StemPro-34 Medium was cultured at 37 ℃ in 5% CO ₂ 、5%O ₂ Culturing for 2 days under the condition;

step 3): on day 2 after electroporation, fresh StemPro ™ chamber-34 medium was replaced; the reprogramming induction medium was replaced every 2 days thereafter and 5% CO at 37 deg.C ₂ 、5%O ₂ Culturing under the condition until cloning occurs;

step 4): induced pluripotent stem cell clone appears 10-12 days after electrotransfer, and reprogramming induction culture is replaced based on 37 ℃ and 5% CO ₂ Continuously culturing under the condition, replacing the culture medium every 1 day during the culture, and calculating the number of the appeared induced pluripotent stem cell clones;

step 5): when the reprogrammed pluripotent stem cell clone grows to be suitable for picking, picking the induced pluripotent stem cell clone and inoculating the induced pluripotent stem cell clone into a new culture plate coated with matrigel at 37 ℃ and 5% CO ₂ And continuously subculturing under the condition, wherein during the culture, the reprogramming induction culture medium is replaced once every 1 day, and the subculturing is performed once every 3 days.

The reprogramming plasmids and reprogramming induction media used in experimental groups 1-3 are shown in Table 1, wherein the plasmid pCXLE-hOCT3/4-shP53 is from Addgene, #27077), and the coding sequence of hOCT3/4 contained in the plasmid is:

ATGGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGTGATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGCTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCTGAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGGCGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAGACCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGGCCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGAAGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAACTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACACAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAAACGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCAGGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAACCAGTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCGAAACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGGATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCAGCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGGACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGGAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCTTTCCCCCTGTCTCCGTCACCACTCTGGGCTCTCCCATGCATTCAAACTGATGATGA（SEQ ID NO:1）。

the sequence of shP53 contained in this plasmid is:

CCGGGTCCAGATGAAGCTCCCAGAACTCGAGTTCTGGGAGCTTCATCTGGACTTTTT（SEQ ID NO:2）。

plasmid pCXLE-sh Cohesin is obtained by integrating the DNA sequence of shRNA of Cohesin into pCXLE (Addgene, # 37626) vector by restriction endonuclease BamHI, the mRNA sequence of Cohesin can be referred to GenBank Accession number XM-011512331.2, and the DNA sequence of shRNA of Cohesin is used as follows:

CACCGCCTTCATGTTGTTTGCATTCCGAAGAATGCAAACAACATGAAGGC（SEQ ID NO:3）。

the components of the experimental reprogramming induction culture solution are as follows: IX-PSC basal medium (containing DMEM/F12, 20. mu.g/mL ascorbic acid, 40ng/mL FGF2, 1ng/mL TGF β 1, 6ng/mL neuregulin 1, 15ng/mL IGF-1, 2.5. mu.g/mL superoxide dismutase, 2.5. mu.g/mL catalase, 5mg/mL human serum albumin, 20. mu.g/mL transferrin, and 20. mu.g/mL sodium selenite), and 2. mu.M CHIR99021, 2. mu.M VPA, 50nM PD0325901, 2.5. mu.M Thiazovin, and 500. times Trace element B, 500. times Trace element C, and 2mM 4-hydroxyethylpiperazine ethanesulfonic acid. Wherein the Trace element B is Trace elements B, manufactured by Corning, with a product number of 25-022; trace elements C is Trace elements C, Corning, product number 25-023. The electrotransformation instrument system is purchased from Saimei Feishale science and technology (China) Co., Ltd. (MPK 5000, MPK 10096), and the electrotransformation conditions are 1650V and 10 ms.

The reprogramming method of Experimental group 4 was that Sendai virus (Gibco, A16517, A16518) was transfected into PBMC cells, and the reprogramming induction medium was a commercially available induced pluripotent Stem cell Medium mTeSR ^TM 1 (manufacturer stemcel Technologies inc., cat 85850), see table 1.

TABLE 1 design of different reprogramming methods

Experimental group	Reprogramming mode	Reprogramming induction medium
			Experimental group 1	pCXLE-hOCT3/4-shP53 and pCXLE-sh Cohesin transfections	mTeSR ^TM 1
Experimental group 2	pCXLE-hOCT3/4-shP53 transfection	Experimental reprogramming induction culture solution
			Experimental group 3	pCXLE-hOCT3/4-shP53 and pCXLE-sh Cohesin transfection	Experimental reprogramming induction culture solution
Experimental group 4	Sendai virus transfection	mTeSR ^TM 1

In the reprogramming process, when cell clones with flat and homogeneous shapes and obvious boundary outlines appear, namely induced pluripotent stem cell clones appear, the counting of the appeared induced pluripotent stem cell clones (hereinafter referred to as clones) of each experimental group is counted, and the effectiveness of the selected plasmids and reagents on reprogramming efficiency is researched.

FIG. 2 shows the results of different experimental groups per 1X 10 ⁶ Clones were generated after reprogramming of individual PBMCs. As can be seen from FIG. 2, the induced pluripotent stem cells obtained from the experimental groups 1-3 were all greater than those obtained from the experimental group 4, whereas the induced pluripotent stem cells obtained from the experimental group 3 were the most when the experimental group 3 was compared with the experimental groups 1 and 2, which indicates that the above-mentioned experimental reprogramming induction culture solution and pCXLE-sh cohesin exert a critical effect on stem cell induction. Comparing the experimental group 3 with the experimental group 4, it can be seen that the experimental group 3 has an average of 15 more clones than the experimental group 4. Currently, the accepted method for reprogramming blood cells is Sendai virus transfection and at mTeSR ^TM 1 to further obtain stem cell clone, mTeSR ^TM 1 is a classical inducerThe experiments prove that the combination of the pCXLE-sh cohesin plasmid and the experimental reprogramming induction culture solution can obtain higher pluripotent stem cell clone yield.

Example 2 identification of the potential of induced pluripotent Stem cells to differentiate into three germ layer tissue

EB differentiation function identification was performed on the induced pluripotent stem cells obtained from step 1) -5) of experiment group 3 in example 1, and the specific steps were as follows: inducing the obtained cells to differentiate into EB by using KOSR EB differentiation complete culture medium, and detecting ectodermal marker protein beta-Tubulin III, mesodermal marker protein SMA and endodermal marker protein AFP by carrying out immunofluorescence staining on the EB.

FIG. 3 shows the results of differentiation identification of the obtained cell line EB, which illustrates the potential of differentiation into tissues of each of the three germ layers of the cell line obtained by the reprogramming method.

Example 3 induced pluripotent Stem cell pluripotent immunofluorescence assay

Cloned cells obtained from induced pluripotent stem cells obtained in experimental group 3 of example 1 through steps 1) -5) were plated with mTeSR in a 4-well plate ^TM 1 complete culture medium culture, observing the cell density and the cell growth state under a 4-fold lens and a 10-fold lens, observing whether the cell is polluted by bacteria and fungi under a 20-fold lens, observing the cell density, if the cell density reaches 30%, carrying out non-pluripotent immunofluorescence detection, and detecting stem cell characteristic factors of Nanog, Oct4, Sox2 and SSEA 4.

FIG. 4 shows the results of the measurement of pluripotent immunofluorescence of the obtained cells, which revealed that the obtained cells expressed the pluripotent genes Nanog, Oct4, Sox2 and SSEA4, and were pluripotent stem cells.

Example 4 flow assay for induced pluripotent Stem cell pluripotency

Using mTeSR ^TM 1 complete culture medium culture of cells obtained from induced pluripotent stem cells obtained from step 1) -5) of the experimental group 3 of example 1, when the cells grow to about 90%, using Accutase to digest the cells, gently transferring the cell suspension into a 15ml centrifuge tube, mixing the cells uniformly, counting the cells, and ensuring that 2 × 10 cells are obtained for each 1 Marker flow ⁶ And (4) cells. Centrifuging to remove supernatant, adding 90% cold into centrifuge tubeThe cells were fixed in methanol and treated for flow detection of surface antigens Nanog, Oct4 and SSEA4 on a flow cytometer.

FIG. 5 shows the flow-through identification results of the obtained cell lines, which indicate that the cell purity of the cells expressing pluripotency genes Nanog, Oct4 and SSEA4 is more than 97%, and thus, the purity of the obtained induced pluripotent stem cells is high.

Example 5 induced pluripotent Stem cell karyotype identification

The method comprises the following specific steps: using mTeSR ^TM 1 complete medium culture of induced pluripotent stem cells obtained from the induced pluripotent stem cells obtained through steps 1) to 5) of experiment group 3 in example 1. The cell number is measured by taking cells in one hole of a 6-hole plate, karyotype analysis can be carried out when the cell density reaches about 70%, 0.2 mu g/ml colchicine is added into each hole, the treatment is carried out for 2 hours, after Accutase digestion, KCL hypotonic is carried out for 30 minutes, and karyotype detection is carried out after the treatment by adding a fixing solution (glacial methanol: acetic acid = 3: 1).

FIG. 6 shows the results of karyotype examination of the obtained cell lines, which revealed that the karyotype analysis of the obtained cell lines was normal.

The present invention can reprogram human induced pluripotent stem cells from peripheral blood mononuclear cells taken from the blood of a patient. The method for inducing the pluripotent stem cells has the advantages that: 1) the materials are easy to obtain, the mononuclear cells of the peripheral blood can be taken from the blood of a patient, the number is not limited, and moral disputes can be avoided; 2) human transcription factor OCT4, p53 gene expression inhibitor and Cohesin gene expression inhibitor are adopted during cell reprogramming, and the needed factors are few; 3) the electrotransfer reprogramming induction culture medium is added with a GSK-3 inhibitor, an acetylase inhibitor, an MEK inhibitor, a ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid, all components play a role in a synergistic manner, and the cell induction reprogramming efficiency is improved while cell growth and proliferation are maintained in the iPS cell reprogramming process; compared with the prior art, the method greatly shortens the induction time, only needs 10 to 12 days, and can be used for large-scale industrial preparation.

Sequence listing

<110> Shanghai Isale Biotech Co., Ltd

<120> System and method for reprogramming peripheral blood mononuclear cells to induced pluripotent stem cells

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cctcctggag ggccaggaat cgggccgggg gttgggccag gctctgaggt gtgggggatt 180

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ggagtggggc tagtgcccca aggcggcttg gagacctctc agcctgaggg cgaagcagga 300

gtcggggtgg agagcaactc cgatggggcc tccccggagc cctgcaccgt cacccctggt 360

gccgtgaagc tggagaagga gaagctggag caaaacccgg aggagtccca ggacatcaaa 420

gctctgcaga aagaactcga gcaatttgcc aagctcctga agcagaagag gatcaccctg 480

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caaacgacca tctgccgctt tgaggctctg cagcttagct tcaagaacat gtgtaagctg 600

cggcccttgc tgcagaagtg ggtggaggaa gctgacaaca atgaaaatct tcaggagata 660

tgcaaagcag aaaccctcgt gcaggcccga aagagaaagc gaaccagtat cgagaaccga 720

gtgagaggca acctggagaa tttgttcctg cagtgcccga aacccacact gcagcagatc 780

agccacatcg cccagcagct tgggctcgag aaggatgtgg tccgagtgtg gttctgtaac 840

cggcgccaga agggcaagcg atcaagcagc gactatgcac aacgagagga ttttgaggct 900

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Claims

1. A reprogramming system for reprogramming Peripheral Blood Mononuclear Cells (PBMCs) into human induced pluripotent stem cells, comprising a reprogramming induction culture solution and one or more vectors capable of expressing human transcription factor OCT4, a p53 gene expression inhibitor and a Cohesin gene expression inhibitor; wherein the reprogramming induction culture fluid comprises IX-PSC basal medium, GSK-3 inhibitor, deacetylase inhibitor, MEK inhibitor, ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid; wherein the IX-PSC basal medium is prepared by taking DMEM/F12 as a basal medium and adding 10-30 mug/mL ascorbic acid, 30-50 ng/mL FGF2, 0.5-2 ng/mL TGF beta 1, 3-9 ng/mL neuregulin 1, 10-20 ng/mL IGF-1, 2-3 mug/mL superoxide dismutase, 2-3 mug/mL catalase, 1-10mg/mL human serum albumin, 10-30 mug/mL transferrin and 10-30 mug/mL sodium selenite;

wherein the inhibitor of p53 gene expression and inhibitor of Cohesin gene expression are inhibitory RNA molecules;

wherein the GSK-3 inhibitor is CHIR99021, the deacetylase inhibitor is VPA, the MEK inhibitor is PD0325901, and the ROCK inhibitor is Thiazovivin;

wherein in the reprogramming induction culture solution, the concentration of the human serum albumin is 1-10mg/mL, the concentration of the GSK-3 inhibitor is 0.5-3 mu M, the concentration of the deacetylase inhibitor is 0.3-3 mu M, the concentration of the MEK inhibitor is 10-100nM, the concentration of the ROCK inhibitor is 0.5-5 mu M, and the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid is 1-10 mM.

2. The reprogramming system of claim 1, wherein the inhibitory RNA molecule is an antisense RNA, miRNA, siRNA or shRNA.

3. The reprogramming system of claim 2, wherein the inhibitory RNA molecule is an shRNA.

4. The reprogramming system of any one of claims 1-3, wherein said one or more vectors are non-genomically integrated vectors.

5. The reprogramming system of claim 4, wherein the one or more vectors are plasmids.

6. The reprogramming system of any one of claims 1 to 3, wherein in the reprogramming induction culture fluid, the human serum albumin concentration is 5mg/mL, the GSK-3 inhibitor concentration is 2 μ M, the deacetylase inhibitor concentration is 2 μ M, the MEK inhibitor concentration is 50nM, the ROCK inhibitor concentration is 2.5 μ M, and the 4-hydroxyethylpiperazine ethanesulfonic acid concentration is 5 mM.

7. A method of reprogramming Peripheral Blood Mononuclear Cells (PBMCs) to induced pluripotent stem cells, the method comprising:

wherein the reprogramming induction culture fluid comprises IX-PSC basal medium, GSK-3 inhibitor, deacetylase inhibitor, MEK inhibitor, ROCK inhibitor and 4-hydroxyethyl piperazine ethanesulfonic acid; wherein the IX-PSC basal medium is prepared by taking DMEM/F12 as a basal medium and adding 10-30 mug/mL ascorbic acid, 30-50 ng/mL FGF2, 0.5-2 ng/mL TGF beta 1, 3-9 ng/mL neuregulin 1, 10-20 ng/mL IGF-1, 2-3 mug/mL superoxide dismutase, 2-3 mug/mL catalase, 1-10mg/mL human serum albumin, 10-30 mug/mL transferrin and 10-30 mug/mL sodium selenite;

8. The method of claim 7, wherein the inhibitory RNA molecule is an antisense RNA, miRNA, siRNA, or shRNA.

9. The method of claim 8, wherein the inhibitory RNA molecule is an shRNA.

10. The method of any one of claims 7-9, wherein the one or more vectors are non-genomically integrated vectors.

11. The method of claim 10, wherein the one or more vectors are plasmids.

12. The method of any one of claims 7-9, wherein the total concentration of the one or more carriers is 1-3 μ g carrier/1.0 x 10 ⁵ Blood cells.

13. The method of claim 12, wherein the total concentration of the one or more carriers is 2 μ g carrier/1.0 x 10 ⁵ Blood cells.

14. The method of any one of claims 7-9, wherein in the reprogramming induction culture fluid, the human serum albumin concentration is 5mg/mL, the GSK-3 inhibitor concentration is 2 μ Μ, the deacetylase inhibitor concentration is 2 μ Μ, the MEK inhibitor concentration is 50nM, the ROCK inhibitor concentration is 2.5 μ Μ, and the 4-hydroxyethylpiperazine ethanesulfonic acid concentration is 5 mM.

15. The method according to any one of claims 7-9, comprising the steps of:

step 1): peripheral Blood Mononuclear Cells (PBMC) are treated at 35-39 ℃ with 3-7% CO ₂ And 0.5 to 2X 10 ⁶ Culturing the cells/well in a well plate for 1-3 days;

step 3): day 2-3 after transfection, fresh StemPro-34 medium was used instead; then replacing the reprogramming induction culture solution every 1-3 days, and carrying out 3-7% CO treatment at the temperature of 35-39 DEG C ₂ And 3 to 7% of O ₂ Until the induction of pluripotent stem cell clone appears.

16. The method of claim 15, comprising the steps of:

step 2): according to a total concentration of 2. mu.g carrier/1.0X 10 ⁵ Blood cells the vector or vectors are transfected into peripheral blood mononuclear cells, which are plated onto tissue culture plates using StemPro-34 medium at 37 ℃ with 5% CO ₂ And 5% of O ₂ Culturing for 2 days;

step 3): day 2 post-transfection, fresh StemPro-chambers-34 were replaced; thereafter, the reprogramming induction medium was replaced every 2 days at 37 ℃ with 5% CO ₂ And 5% of O ₂ Until the induction of pluripotent stem cell clone appears.

17. The method of claim 16, the method further comprising: step 4): replacing the reprogramming induction culture solution after the induced pluripotent stem cell clone appears, and carrying out the reprogramming induction culture solution at 35-39 ℃ and 3-7% CO ₂ The culture was continued with replacement of the stem cell medium every 1 to 2 days during the culture.

18. A kit comprising the reprogramming system of any one of claims 1 to 6.

19. Use of the reprogramming system of any one of claims 1 to 6 or the kit of claim 18 for inducing reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) into human induced pluripotent stem cells.