Induced pluripotent stem cell and preparation method and application thereof

Abstract

The present invention discloses an induced pluripotent stem cell and a preparation method and application thereof. A non-integrating plasmid is used to perform cell nucleus transfection on CD34+ cells, and after transfection, the cells are cultured and passaged in a reprogramming complete medium, wherein the reprogramming complete medium contains placental polypeptide, lercanidipine and dehydrogenated echinopsine. The present invention uses a non-integrating plasmid to reprogram CD34+ cells of human origin, and successfully induces the formation of plasmid non-integrating induced pluripotent stem cells. The generated induced pluripotent stem cells have no exogenous genes and no virus insertion, thus avoiding the risk of gene integration, and have high efficiency and safety. Placental active polypeptide, lercanidipine and dehydrogenated echinopsine are added during the preparation of induced pluripotent stem cells, which greatly improves the cloning rate of CD34+ induced pluripotent stem cells derived from human peripheral blood and the accuracy of cell karyotype.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an induced pluripotent stem cell and a preparation method and application thereof. Background Art

[0002] With the development of cell research, adult cells can change their gene expression profiles through reprogramming and regain pluripotency. Early methods of somatic cell reprogramming mainly include somatic cell nuclear transfer and cell fusion, but these two methods still have problems such as developmental abnormalities, immune deficiencies and ethical issues. In 2006, Takahashi and Yamanaka screened out a combination of four transcription factors OSKM: Oct4, Sox2, Klf4, and c-Myc, which can reprogram terminally differentiated somatic cells into iPSCs (induced pluripotent stem cells) with embryonic stem cell characteristics, greatly expanding the application potential of stem cell technology in clinical medicine. Now, the research and application system of iPSCs is becoming more and more mature, but it also faces defects such as low reprogramming efficiency, unclear reprogramming mechanism, strong heterogeneity of reprogrammed cells and safety issues.

[0003] At present, many laboratories around the world have developed a variety of reprogramming methods, which can be roughly divided into the following four categories based on the classification of reprogramming vectors: (1) Viral method: This includes random genome insertion viral vectors (lentivirus) and non-genomic insertion vectors (Sendai virus). The disadvantage of this method is the potential risk brought by random genome insertion or the instability caused by the lengthy virus dilution process. (2) DNA method: This includes a variety of plasmid types such as Piggy Bac, Sleeping Beaμty and Episomal. This method is characterized by low reprogramming efficiency. (3) RNA method and protein method: This method has a complicated operation process and poor practicality in industrial application. (4) Small molecule compound method: This is a reprogramming method that is currently being used more and more widely, but there are large individual differences in its reprogramming efficiency.

[0004] However, the first three methods currently use reprogramming culture media containing serum components during the induction process, such as using knockout serum in the presence of a feeder layer or using culture media containing N2 or B27 additives in the absence of a feeder layer, and these culture media also contain serum proteins including transferrin or other animal-derived components. The presence of serum protein components causes inconvenience in the clinical application of induced pluripotent stem cells, which is not conducive to the industrial induction of reprogramming and the formulation of standardized processes. The small molecule method still needs to solve the problem of large individual differences, that is, how to achieve approximately equivalent reprogramming efficiency in multiple cell types is still a major problem in the field of regenerative medicine.

[0005] Therefore, there is an urgent need to provide an efficient, safe and simple method for expanding induced pluripotent stem cells, avoiding the risk of gene integration and greatly improving the cloning rate of induced pluripotent stem cells. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides an induced pluripotent stem cell and a preparation method and application thereof, which solves the problems of poor safety, low reprogramming efficiency, complex operation and the like in the existing methods for preparing induced pluripotent stem cells. Human-derived CD34+ cells are reprogrammed using a non-integrating plasmid to successfully induce the formation of plasmid-non-integrating induced pluripotent stem cells, and the resulting induced pluripotent stem cell clone formation rate and cell karyotype accuracy are significantly improved.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing induced pluripotent stem cells, the method comprising: performing nucleofection on CD34+ cells using a non-integrating plasmid, and culturing and passaging the cells in a complete reprogramming medium after transfection, wherein the complete reprogramming medium contains placental polypeptide, lercanidipine and dehydrogenated echinopsine.

[0009] The present invention uses a non-integrating plasmid to reprogram CD34+ cells from human sources, successfully inducing the formation of plasmid non-integrating induced pluripotent stem cells, the generated induced pluripotent stem cells have no exogenous genes and no virus insertion, thus avoiding the risk of gene integration, having high efficiency and safety, and the method is simple and repeatable, and placental active polypeptide, lercanidipine and harmine (a specific tyrosine phosphorylation-regulated kinase inhibitor) are added during the preparation of the induced pluripotent stem cells, and the addition ratio of the placental active polypeptide, lercanidipine and harmine is accurately controlled, thereby greatly improving the cloning rate of induced pluripotent stem cells of CD34+ cells derived from human peripheral blood and the accuracy of cell karyotype.

[0010] Preferably, the mass ratio of the placental polypeptide, lercanidipine and harmine is (2-10):5:5.

[0011] The specific point values ​​in the above 2-10 can be selected as 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0012] Preferably, the mononuclear cells are derived from any one of humans, rats, mice, cows or pigs.

[0013] Preferably, the use of a non-integrating plasmid to perform nuclear transfection on CD34+ cells comprises introducing the reprogramming gene into CD34+ hematopoietic stem cells using a non-integrating episome vector.

[0014] Preferably, the reprogramming gene includes any one of OCT4, SOX2, NANOG or SSEA-1, or a combination of at least two of them.

[0015] Preferably, the non-integrating episomal vector comprises an Episomal plasmid vector.

[0016] Preferably, the culture time is 20-30 days.

[0017] Preferably, the subculturing is performed when the cell confluence reaches 80%-90%, and the number of subculturing generations is 8-10 generations.

[0018] The specific point values ​​in the above 20-30 days can be selected as 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, etc.

[0019] The specific point values ​​among the above 80%-90% can be selected as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% and the like.

[0020] The specific point values ​​in the above 8-10 generations can be selected from 8th generation, 9th generation, 10th generation, etc.

[0021] In a second aspect, the present invention provides an induced pluripotent stem cell, wherein the induced pluripotent stem cell is prepared by the method for preparing induced pluripotent stem cells described in the first aspect.

[0022] In a third aspect, the present invention provides a pharmaceutical composition comprising the induced pluripotent stem cells described in the second aspect.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses non-integrating plasmids to reprogram human CD34+ cells and successfully induces the formation of plasmid non-integrating induced pluripotent stem cells. The resulting induced pluripotent stem cells have no exogenous genes and no virus insertion, avoiding the risk of gene integration, and are highly efficient and safe. The method is simple and reproducible, laying a foundation for the next step of establishing more research on induced pluripotent stem cells for blood system diseases;

[0025] (2) In the process of preparing induced pluripotent stem cells by the method of the present invention, placental active polypeptide, lercanidipine and dehydrogenated echinopsine are added, and the addition ratio of placental active polypeptide, lercanidipine and dehydrogenated echinopsine is precisely controlled, which greatly improves the cloning rate of induced pluripotent stem cells of CD34+ cells derived from human peripheral blood and the accuracy of cell karyotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of the induced pluripotent stem cells prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0028] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0029] Placental peptides were purchased from Guizhou Taibang Biological Products Co., Ltd., with approval number: National Medicine Standard H20046260; non-integrating plasmid Epi5TM Episomal iPSC Reprogramming Kit (Thermo Fisher, catalog number: A15960); alkaline phosphatase colorimetric kit was purchased from Dalian Meilun Biotechnology Co., Ltd., catalog number: MA0197; CellAdhere TM Laminin-521 (Stem Cell, Catalog No.: 200-0117); ReproTeSR TM Reprogramming complete medium (Stem Cell, Cat. No. 05926). TM (Stem Cell Company, Catalog No.: 05872). Lercanidipine (MCE Company, Catalog No.: HY-B0612), Harmine (MCE Company, Catalog No.: HY-B0464A).

[0030] Example 1

[0031] Preparation of induced pluripotent stem cells.

[0032] (1) Isolation and culture of human CD34+ cells

[0033] Human cord blood mononuclear cells (MNC) were isolated by Ficoll-Paque Premium (P=1.077) density gradient centrifugation. After obtaining MNC, CD34+ cells were sorted using Human CD34 MicroBead kit. 8 Add 100 μL Fc-R blocking agent to each 10 cells. 8 Add 100 μL CD34+ Microbeads to the cells, mix thoroughly, incubate at 4°C in the dark for 30 min, and sort CD34+ cells using MACS Separation Columns. TM SFEM Il (Article No. 9605).

[0034] (2) Treatment of induced pluripotent stem cells with placental peptides, lercanidipine, and dehydrogenated echinopsine

[0035] CD34+ cells were cultured for 9 days and then nuclear transfection was performed using an Amaxa nuclear transfection instrument (i.e., transfection day 0). The non-integrated plasmid Epi5 TM Episomal iPSC Reprogramming Kit (Cat. No. A15960), plasmid transfection program T-016, transfected cells (2×10 6 Cells were seeded into 12-well plates and cultured 24 hours later, and then transferred to the wells pre-coated with CellAdhere TM Laminin-521 (Cat. No. 200-0117) was added to each well of a 6-well plate. TM 2 mL of complete reprogramming medium (Cat. No. 05926) was added with a mixture of placental peptides, lercanidipine, and dehydrogenated echinopsine in a ratio of 6:5:5. TM The reprogramming complete medium was replaced every day, and clones were picked for subculture on the 15th day after electroporation. Small molecule compounds lercanidipine and dehydrogenated echinopsine solution preparation: 10 μL 10mM concentration lercanidipine was dissolved in 10mL ReproTeSR TM Reprogramming complete medium to obtain a working solution with a concentration of 10 μM. Add to complete medium at a volume ratio of 2.5% to a final concentration of 250 nM.

[0036] (3) Passaging and expansion of induced pluripotent stem cells

[0037] Observe the cell confluence and subculture when the confluence reaches 90%. Add 1 mL of ReLeSR TM, incubate at 37°C for 7 min. Use a 5 mL pipette to transfer the detached cell aggregates to a 15 mL centrifuge tube. The size of the cell aggregates is 50-200 μm, which is the appropriate aggregate size for plating. TM Plus culture wells. When a colony density reaches 500 single cells, the culture is distributed at a ratio of 1:10 (i.e., the cell aggregates in one culture well are plated in 10 new culture wells), and the plate is placed in a 37°C incubator. Subculture is performed every 7 days. TM Plus, change the medium every day, evaluate the culture, and monitor the growth status until the next subculture. Subculture for 8-10 generations until the cell line is basically stable.

[0038] Example 2

[0039] Preparation of induced pluripotent stem cells.

[0040] (1) Isolation and culture of human CD34+ cells

[0041] Human cord blood mononuclear cells (MNC) were isolated by Ficoll-Paque Premium (P=1.077) density gradient centrifugation. After obtaining MNC, CD34+ cells were sorted using Human CD34 MicroBead kit. 8 Add 100 μL Fc-R blocking agent to each 10 cells. 8 Add 100 μL CD34+ Microbeads to the cells, mix thoroughly, incubate at 4°C in the dark for 30 min, and sort CD34+ cells using MACS Separation Columns. TM SFEM Il (Article No. 9605).

[0042] (2) Treatment of induced pluripotent stem cells with placental peptides, lercanidipine, and dehydrogenated echinopsine

[0043] CD34+ cells were cultured for 9 days and then nuclear transfection was performed using an Amaxa nuclear transfection instrument (i.e., transfection day 0). The non-integrated plasmid Epi5 TM Episomal iPSC Reprogramming Kit (Cat. No. A15960), plasmid transfection program T-016, transfected cells (2×10 6 Cells were seeded into 12-well plates and cultured 24 hours later, and then transferred to the wells pre-coated with CellAdhere TMLaminin-521 (Cat. No. 200-0117) was added to each well of a 6-well plate. TM 2 mL of complete reprogramming medium (Cat. No. 05926) was added with a mixture of placental peptides, lercanidipine, and dehydrogenated echinopsine at a ratio of 2:5:5. TM The reprogramming complete medium was replaced every day, and clones were picked for subculture on the 15th day after electroporation.

[0044] (3) Passaging and expansion of induced pluripotent stem cells

[0045] Observe the cell confluence and subculture when the confluence reaches 90%. Add 1 mL of ReLeSR TM , incubate at 37°C for 7 min. Use a 5 mL pipette to transfer the detached cell aggregates to a 15 mL centrifuge tube. The size of the cell aggregates is 50-200 μm, which is the appropriate aggregate size for plating. TM Plus culture wells. When the colony density is 500, one colony contains about 500 single cells. The culture is distributed at a ratio of 1:10 (i.e., the cell aggregates in one culture well are plated in 10 new culture wells), and the plate is placed in a 37°C incubator. Subculture is performed every 7 days. TM Plus, change the medium every day, evaluate the culture, and monitor the growth status until the next subculture. Subculture for 8-10 generations until the cell line is basically stable.

[0046] Example 3

[0047] Preparation of induced pluripotent stem cells.

[0048] (1) Isolation and culture of human CD34+ cells

[0049] Human cord blood mononuclear cells (MNC) were isolated by Ficoll-Paque Premium (P=1.077) density gradient centrifugation. After obtaining MNC, CD34+ cells were sorted using Human CD34 MicroBead kit. 8 Add 100 μL Fc-R blocking agent to each 10 cells. 8 Add 100 μL CD34+ Microbeads to the cells, mix thoroughly, incubate at 4°C in the dark for 30 min, and sort CD34+ cells using MACS Separation Columns. TMSFEM Il (Article No. 9605).

[0050] (2) Treatment of induced pluripotent stem cells with placental peptides, lercanidipine, and dehydrogenated echinopsine

[0051] CD34+ cells were cultured for 9 days and then nuclear transfection was performed using an Amaxa nuclear transfection instrument (i.e., transfection day 0). The non-integrated plasmid Epi5 TM Episomal iPSC Reprogramming Kit (Cat. No. A15960), plasmid transfection program T-016, transfected cells (2×10 6 Cells were seeded into 12-well plates and cultured 24 hours later, and then transferred to the wells pre-coated with CellAdhere TM Laminin-521 (Cat. No. 200-0117) was added to each well of a 6-well plate. TM 2 mL of complete reprogramming medium (Cat. No. 05926) was added to the mixture of placental peptides, lercanidipine and dehydrogenated echinopsine at a ratio of 2:1:1. TM The reprogramming complete medium was replaced every day, and clones were picked for subculture on the 20th day after electroporation.

[0052] (3) Passaging and expansion of induced pluripotent stem cells

[0053] Observe the cell confluence and subculture when the confluence reaches 90%. Add 1 mL of ReLeSR TM , incubate at 37°C for 7 min. Use a 5 mL pipette to transfer the detached cell aggregates to a 15 mL centrifuge tube. The size of the cell aggregates is 50-200 μm, which is the appropriate aggregate size for plating. TM Plus culture wells. When the colony density is 500, one colony contains about 500 cells. The culture is distributed at a ratio of 1:100 (i.e., the cell aggregates in one culture well are plated in 100 new culture wells), and the plate is placed in a 37°C incubator. Subculture is performed every 7 days. TM Plus, change the medium every day, evaluate the culture, and monitor the growth status until the next subculture. Subculture for 8-10 generations until the cell line is basically stable.

[0054] Comparative Example 1

[0055] The only difference from Example 1 is that the complete reprogramming medium does not contain placental polypeptide, and its weight is proportionally distributed to the weight of lercanidipine and dehydrogenated echinopsine.

[0056] Comparative Example 2

[0057] The only difference from Example 1 is that the complete reprogramming medium does not contain lercanidipine, and its weight is proportionally distributed to the weight of placental polypeptide and dehydrogenated echinopsine.

[0058] Comparative Example 3

[0059] The only difference from Example 1 is that the complete reprogramming medium does not contain dehydrogenated harmine, and its weight is proportionally distributed to the weight of the placental polypeptide and lercanidipine.

[0060] Comparative Example 4

[0061] The only difference from Example 1 is that the complete reprogramming medium does not contain placental polypeptide and lercanidipine, and their weight portions are proportionally distributed to the weight of dehydrogenated echinopsine.

[0062] Comparative Example 5

[0063] The only difference from Example 1 is that the complete reprogramming medium does not contain placental polypeptide and dehydrogenated echinopsine, and their weight portions are proportionally distributed to the weight of lercanidipine.

[0064] Comparative Example 6

[0065] The only difference from Example 1 is that the complete reprogramming medium does not contain placental polypeptide, lercanidipine and dehydrogenated echinopsine.

[0066] Comparative Example 7

[0067] The only difference from Example 1 is that the mass ratios of placental polypeptide, lercanidipine and dehydrogenated echinopsine are 8:1:1 respectively.

[0068] Comparative Example 8

[0069] The only difference from Example 1 is that the mass ratio of placental polypeptide, lercanidipine and dehydrogenated echinopsine is 1:6:6.

[0070] Test Example 1

[0071] Karyotyping of induced pluripotent stem cells.

[0072] Karyotype analysis was performed by karyotype banding: the P8 cells prepared in Examples 1-3 and Comparative Examples 1-8 were transferred to a 6-well plate paved with Laminin-521, cultured for 2 days, and treated with 0.25 g / mL colchicine for 4 hours. Each cell line was digested with 500 μL of 0.05% pancreatin, neutralized by adding mTeSRTMPlus, centrifuged at 200 × g for 5 minutes, the supernatant was discarded, the cells were harvested, and 1 mL of hypotonic solution (0.4% sodium citrate and 0.4% KCl were prepared in a ratio of 1:1) was added, 37°C, 5 minutes. 350 μL of fixative (methanol and glacial acetic acid volume ratio of 3:1) was added, and centrifuged at 200 × g for 5 minutes. The supernatant was removed, and 4 mL of fixative (methanol and glacial acetic acid volume ratio of 3:1) was added, and fixed for 40 minutes. Centrifuge at 200×g for 5 minutes, remove the supernatant, add 2mL of fixative (methanol and glacial acetic acid volume ratio is 3:1) again, and fix for 20 minutes. Centrifuge at 200×g for 5 minutes, remove the supernatant, add 200μL of fresh fixative (methanol and glacial acetic acid volume ratio is 3:1), and then drop 3 slides, dry, and place in a 65°C oven overnight. On the second day, place the slide in 0.25% trypsin for 30 seconds and in a 37°C water bath for 30 minutes. Add 1 drop of Giemsa stock solution to the slide for 15 seconds, add 2 drops of phosphate buffer and blow evenly, stain at 25°C for 10 minutes, gently wash with running water, and dry at 25°C. After Gemisa staining, 30 cells in the metaphase division phase were randomly selected for observation and the karyotype accuracy was statistically analyzed. The results are shown in Table 1. The karyotype of the induced pluripotent stem cells of the present invention is 46XY.

[0073] Table 1

[0074]

[0075]

[0076] As shown in Table 1, the proportion of induced pluripotent stem cells with 46 chromosomes prepared by Examples 1-3 of the present invention is 80%-85%, and the karyotype is the same as the karyotype of normal cells, i.e. (46, XY), indicating that the karyotype of the reprogrammed induced pluripotent stem cells is normal. The proportion of induced pluripotent stem cells with 46 chromosomes prepared by Comparative Examples 1-6 is 70%-78%, and the karyotype is normal, indicating that placental active polypeptide, lercanidipine and B have synergistic effects in improving the correct function of the karyotype of induced pluripotent stem cells. The proportion of induced pluripotent stem cells with 46 chromosomes prepared by Comparative Examples 7-8 is 75% and 76%, and the karyotype is normal, indicating that the precise control of the ratio of placental active polypeptide, lercanidipine and dehydrogenated echinopsine can increase the proportion of normal karyotypes of induced pluripotent stem cells.

[0077] Test Example 2

[0078] Alkaline phosphatase detection: Fix the P10 cells prepared in Examples 1-3 and Comparative Examples 1-8 with 4% paraformaldehyde at 25°C for 5 minutes, discard the paraformaldehyde fixative, wash twice with TBST buffer, add AP buffer, balance at 25°C for 6 minutes, add BCIP / NBT colorimetric solution in the dark, color development time 25 minutes, scan and take pictures, count the number of positive clones, and calculate the reprogramming efficiency. Reprogramming efficiency (%) = (number of positive clones / number of cells in electrotransfection reaction) × 100%. The results are shown in Table 2.

[0079] Table 2

[0080] Group Number of positive clones <![CDATA[Cell number (105)]]> Cloning efficiency Example 1 500 1 0.5％ Example 2 460 1 0.46％ Example 3 490 1 0.49％ Comparative Example 1 401 1 0.4％ Comparative Example 2 409 1 0.41％ Comparative Example 3 410 1 0.41％ Comparative Example 4 415 1 0.42％ Comparative Example 5 423 1 0.423％ Comparative Example 6 429 1 0.43％ Comparative Example 7 427 1 0.427％ Comparative Example 8 413 1 0.413％

[0081] It can be seen from the results that the cloning efficiency of the induced pluripotent stem cells prepared by Examples 1-3 of the method of the present invention is all above 0.45%. This shows that the induced pluripotent stem cells prepared by the method of the present invention are highly efficient. The cloning efficiency of the induced pluripotent stem cells prepared by Comparative Examples 1-6 is 0.4%, indicating that placental active polypeptide, lercanidipine and harmine have synergistic effects in improving the cloning efficiency of the induced pluripotent stem cell karyotype. The cloning efficiency of the induced pluripotent stem cells prepared by Comparative Examples 7-8 is 0.4%, indicating that precise control of the ratio of placental active polypeptide, lercanidipine and harmine can improve the induction efficiency of CD34+ hematopoietic stem cells induced pluripotent stem cells.

[0082] Test Example 3

[0083] Flow cytometry phenotyping.

[0084] (1) Digestion: Discard the 10th generation cell culture medium prepared in Examples 1-3 and Comparative Examples 1-8, and digest with 0.25% Trypsin. After the cells are digested, add 2 times the volume of cell basal culture medium to terminate the process. Collect the digested cells in a centrifuge tube, centrifuge at 25°C (1000 r / min, 5 min), and discard the supernatant.

[0085] (2) Flow cytometry phenotype detection was performed according to the instructions of the pluripotent stem cell flow cytometry kit (company: R&D Systems, catalog number: FMC001).

[0086] (3) Flow cytometry was used to analyze cell subpopulations. The control tube was tested first, the voltage was adjusted to determine the negative zone, and then the experimental tube was tested. Cells that can bind to the fluorescently labeled antibody will emit corresponding fluorescence, and the cells in the positive zone can be sorted out accordingly. The results of flow cytometry phenotyping are shown in Table 3.

[0087] Table 3

[0088]

[0089]

[0090] From the results, it can be seen that the expression ratios of the pluripotency marker genes OCT4 and SOX2 of the induced pluripotent stem cells prepared by Examples 1-3 of the method of the present invention are all above 90%, the expression ratio of SSEA-4 is above 80%, and the expression ratio of the differentiation marker gene SSEA-1 is below 1%. It shows that the induced pluripotent stem cells prepared by the method of the present invention meet the standards. The results of Comparative Examples 1-6 show that placental active polypeptide, lercanidipine and dehydrogenated echinopsine have synergistic effects in improving the flow phenotype of induced pluripotent stem cells. The results of Comparative Examples 7-8 show that precise control of the ratio of placental active polypeptide, lercanidipine and dehydrogenated echinopsine can provide the flow phenotype of induced pluripotent stem cells and increase the ratio of induced pluripotent stem cells.

[0091] In summary, the present invention uses a non-integrating plasmid to reprogram CD34+ cells from human sources, successfully inducing the formation of plasmid non-integrating induced pluripotent stem cells, the generated induced pluripotent stem cells have no exogenous genes and no virus insertion, avoiding the risk of gene integration, and have high efficiency and safety. The method is simple and repeatable. Placental active polypeptide, lercanidipine and dehydrogenated echinopsine are added during the preparation of induced pluripotent stem cells, and the addition ratio of placental active polypeptide, lercanidipine and dehydrogenated echinopsine is accurately controlled, which greatly improves the cloning rate of CD34+ induced pluripotent stem cells derived from human peripheral blood and the accuracy of cell karyotype.

[0092] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing induced pluripotent stem cells, characterized in that: The method comprises: CD34+ cells were transfected with a non-integrating plasmid, and after transfection, they were cultured and passaged in a complete reprogramming medium, wherein the complete reprogramming medium contained placental polypeptide, lercanidipine and harmine; the mass ratio of the placental polypeptide, lercanidipine and harmine was (2-10):5:5; the final concentration of lercanidipine was 250 nM, and the final concentration of harmine was 720 nM.

2. The method according to claim 1, characterized in that The source of mononuclear cells includes any one of human, rat, mouse, cow or pig.

3. The method according to claim 1, characterized in that The method of using a non-integrating plasmid to perform cell nucleus transfection on CD34+ cells includes introducing the reprogramming gene into CD34+ hematopoietic stem cells using a non-integrating episome vector.

4. The method according to claim 3, characterized in that The reprogramming gene includes any one of OCT4, SOX2, NANOG or SSEA-1 or a combination of at least two of them.

5. The method according to claim 3, characterized in that: The non-integrating episome vector includes an Episomal plasmid vector.

6. The method according to claim 1, characterized in that The culture time is 20-30 days.

7. The method according to claim 1, characterized in that The cell culture is performed when the cell confluence reaches 80%-90%, and the number of generations of the culture is 8-10.

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