CN118272437A - Induced pluripotent stem cell, and preparation method and application thereof - Google Patents
Induced pluripotent stem cell, and preparation method and application thereof Download PDFInfo
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- CN118272437A CN118272437A CN202410432590.9A CN202410432590A CN118272437A CN 118272437 A CN118272437 A CN 118272437A CN 202410432590 A CN202410432590 A CN 202410432590A CN 118272437 A CN118272437 A CN 118272437A
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Abstract
The invention discloses an induced pluripotent stem cell, a preparation method and application thereof. The CD34+ cells were subjected to nuclear transfection using a non-integrative plasmid, and after transfection, were cultured and passaged in a reprogramming complete medium containing placental polypeptide, lercanidipine, and harmine. The invention uses non-integrated plasmid to reprogram human-derived CD34+ cells, successfully induces and forms plasmid non-integrated induced pluripotent stem cells, and the induced pluripotent stem cells have no exogenous genes and no virus insertion, avoid the danger of gene integration, have high efficiency and safety, and greatly improve the cloning formation rate and the correct cell nucleus type of the induced pluripotent stem cells of CD34+ derived from human peripheral blood by adding placenta active polypeptide, lercanidipine and harmine in the process of preparing the induced pluripotent stem cells.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an induced pluripotent stem cell, a preparation method and application thereof.
Background
With the development of cellular research, adult cells can be reprogrammed to alter gene expression profiles to regain multipotent stem. The early somatic reprogramming method mainly comprises somatic cell nuclear transplantation and cell fusion, but the two methods still have the problems of dysplasia, immunodeficiency, ethical problems and the like. In 2006, takahashi and Yamanaka screened 4 transcription factors OSKM: the combination of Oct4, sox2, klf4 and c-Myc can reprogram terminally differentiated somatic cells into iPSCs (induced pluripotent stem cells) with embryonic stem cell characteristics, and greatly expands the application potential of stem cell technology in clinical medicine. At present, research and application systems of iPSCs are mature, but the defects of low reprogramming efficiency, unclear reprogramming mechanism, strong heterogeneity of reprogrammed cells, safety problem and the like are faced.
Currently, a number of reprogramming methods have been developed in a number of laboratories internationally, and can be broadly classified into the following four categories according to the reprogramming vector classification: (1) viral method: these include genome random insertion viral vectors (lentiviruses) and non-genome insertion vectors (Sendai virus), which have the disadvantage of potential risks associated with random insertion of the genome or the invariance of lengthy viral dilution procedures. (2) DNA method: among the various plasmid types, including Piggy Bac, sleeping Bea μty, and Episomal, this type of approach is characterized by low reprogramming efficiency. (3) RNA method and protein method: the method has complex operation process and poor practicability in industrial application. (4) Small molecule method of Compound: this is a reprogramming method which is increasingly widely used at present, but there is a large individual difference in reprogramming efficiency.
However, the current 3 methods all use reprogramming media containing serum components during induction, such as knockout serum in the presence of a feeder layer or medium containing N2 or B27 additives in the absence of a feeder layer, and these media also contain serum proteins including transferrin or other animal-derived components. Due to the existence of serum protein components, the induction of the pluripotent stem cells is inconvenient in the clinical application process, and the preparation of the industrial induction and standardization process of reprogramming is not facilitated. However, the compound small molecule method still needs to solve the problem of large individual difference, namely how to realize approximately equivalent reprogramming Cheng Xiaolv in various cell types is still a great difficulty in the field of regenerative medicine.
Therefore, there is a need to provide a method for efficiently, safely and simply expanding induced pluripotent stem cells, avoiding the risk of gene integration, and greatly improving the cloning efficiency of induced pluripotent stem cells.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides an induced pluripotent stem cell, a preparation method and application thereof, which solve the problems of poor safety, low reprogramming efficiency, complex operation and the like of the existing method for preparing the induced pluripotent stem cell, and the induced pluripotent stem cell is successfully induced by using a CD34+ cell derived from a non-integrated plasmid reprogramming human source, so that the clone formation rate and the accuracy of cell nucleus type of the obtained induced pluripotent stem cell are obviously improved.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a method of preparing an induced pluripotent stem cell, the method comprising: the CD34+ cells were subjected to nuclear transfection using a non-integrative plasmid, and after transfection, were cultured and passaged in a reprogramming complete medium containing placental polypeptide, lercanidipine, and harmine.
The invention uses non-integrated plasmid to reprogram human CD34+ cells, successfully induces and forms plasmid non-integrated induced pluripotent stem cells, and the induced pluripotent stem cells have no exogenous genes and no virus insertion, avoid the danger of gene integration, have high efficiency and safety, have simplicity and repeatability, add placenta active polypeptide, lercanidipine (LERCANIDIPINE) and dehydropeganine (specific tyrosine phosphorylation regulatory kinase inhibitor) in the process of preparing the induced pluripotent stem cells, accurately control the addition proportion of placenta active polypeptide, lercanidipine and dehydropeganine, and greatly improve the cloning formation rate of the induced pluripotent stem cells of human peripheral blood-derived CD34+ cells and the accuracy of cell karyotype.
Preferably, the mass ratio of placenta polypeptide, lercanidipine and harmine is (2-10): 5:5.
Specific point values in the above 2 to 10 may be selected from 2,3, 4, 5, 6, 7, 8, 9, 10, etc.
Preferably, the source of the mononuclear cells comprises any one of human, rat, mouse, bovine or porcine.
Preferably, the nuclear transfection of cd34+ cells using a non-integrating plasmid comprises introducing a reprogramming gene into cd34+ hematopoietic stem cells using a non-integrating adherent vector.
Preferably, the reprogramming genes comprise any one or a combination of at least two of OCT4, SOX2, NANOG, or SSEA-1.
Preferably, the non-integrative adherent vector comprises a Episomal plasmid vector.
Preferably, the incubation is for a period of 20-30 days.
Preferably, the passage is communicated when the cell confluency reaches 80% -90%, and the passage generation is 8-10 passages.
The specific point values of the above 20-30 days can be selected from 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, etc.
Specific point values in the above 80% -90% may be selected from 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% and the like.
Specific point values in the 8-10 generations can be selected from 8 generations, 9 generations, 10 generations and the like.
In a second aspect, the present invention provides an induced pluripotent stem cell prepared by the method for preparing an induced pluripotent stem cell according to the first aspect.
In a third aspect, the invention provides a pharmaceutical composition comprising the induced pluripotent stem cells of the second aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention uses non-integrated plasmid to reprogram human-derived CD34+ cells, successfully induces and forms plasmid non-integrated induced pluripotent stem cells, and the induced pluripotent stem cells have no exogenous genes and no virus insertion, avoid the danger of gene integration, have high efficiency and safety, have simplicity and repeatability, and lay the foundation for the next step of establishing more blood system disease induced pluripotent stem cells;
(2) In the preparation process of the induced pluripotent stem cells, the placenta-active polypeptide, lercanidipine and harmine are added, the addition proportion of the placenta-active polypeptide, lercanidipine and harmine is precisely controlled, and the cloning formation rate of the induced pluripotent stem cells of CD34+ cells from human peripheral blood and the accuracy rate of cell nucleuses are greatly improved.
Drawings
FIG. 1 is a diagram of induced pluripotent stem cells prepared in example 1 of the present invention.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Placental polypeptides were purchased from tabang biologicals, inc. Guizhou, approval document: chinese medicine standard character H20046260; non-integrative plasmid Epi5TM Episomal iPSC Reprogramming Kit (Thermo Fisher Co., ltd.; cat# A15960); alkaline phosphatase chromogenic kit was purchased from Dalian Mei Lun Biotechnology Co., ltd., product number: MA0197; CELLADHERE TM Lamin-521 (Stem Cell Co., ltd.: 200-0117); reproTeSR TM reprogramming complete Medium (Stem Cell Co., ltd.; cat# 05926). ReLeSR TM (Stem Cell Co., ltd.; product number: 05872). Lercanidipine (MCE company, cat# HY-B0612), harmine (MCE company, cat# HY-B0464A).
Example 1
Preparing induced pluripotent stem cells.
(1) Isolated culture of human CD34+ cells
Human peripheral blood mononuclear cells (human cordbloodmononuclear cells, MNC) were isolated by Ficoll-Paque Premium (p=1.077) density gradient centrifugation. After MNC was obtained, cd34+ cells were sorted using a Human CD34MicroBead kit, 100 μl of cd34+ Microbeads was added per 10 8 cells, 100 μl of LFc-R blocker was added per 10 8 cells, and after thoroughly mixing, cd34+ cells were sorted by magnetic column (MACS Separation Columns) by incubation at 4 ℃ for 30min in the absence of light. CD34+ cells were cultured in 6-well plates in StemSpan TM SFEM Il (cat# 9605).
(2) Placental polypeptide, lercanidipine, and harmine-depleted induced pluripotent stem cells
CD34+ cells were cultured for 9 days, and subjected to nuclear transfection (i.e., day 0 transfection) using an Amaxa nuclear transfecter, non-integrated plasmid Epi5 TM Episomal iPSC Reprogramming Kit (cat No. A15960), plasmid transfection program T-016, transfected cells (2X 10 6 cells) were seeded into 12-well plates, transferred 24 hours later to 6-well plates pre-plated with CELLADHERE TM Laminin-521 (cat No. 200-0117), each well was added with 2mL of ReproTeSR TM reprogramming complete medium (cat No. 05926) with the addition of a mixture of placental polypeptide, lercanidipine and harmine, in a ratio of 6:5:5, and clones were picked up for subculture on day 15 after electrotransfection using ReproTeSR TM reprogramming complete medium. The preparation method comprises the following steps of (1) preparing a small molecular compound lercanidipine and harmalane solution: lercanidipine at a concentration of 10. Mu.L and 10mM was dissolved in 10mL ReproTeSR TM reprogramming complete medium to give a working solution at a concentration of 10. Mu.M. Added to the complete medium at a volume ratio of 2.5% at a final concentration of 250nM.
(3) Passage and expansion of induced pluripotent stem cells
Cells were observed for confluence, and passaged at 90%. 1mL ReLeSR TM was added and incubated at 37℃for 7min. The detached cell aggregates were transferred to a 15mL centrifuge tube with a 5mL pipette, the size of the cell aggregates being 50-200 μm, which is the aggregate size suitable for plating. Aggregates were plated into culture wells coated and containing mTeSR TM Plus. When one colony density was 500 single cells, cultures were plated in 10-1 (i.e., 1 culture well cell aggregates were plated into 10 new culture wells, respectively) and the well plates were placed in a 37℃incubator. Passaging was performed every 7 days. Cultures were assessed daily with mTESR TM Plus and growth status monitored until the next passage, passing 8-10 passages until the cell line was essentially stable.
Example 2
Preparing induced pluripotent stem cells.
(1) Isolated culture of human CD34+ cells
Human peripheral blood mononuclear cells (human cordbloodmononuclear cells, MNC) were isolated by Ficoll-Paque Premium (p=1.077) density gradient centrifugation. After MNC was obtained, cd34+ cells were sorted using a Human CD34MicroBead kit, 100 μl of cd34+ Microbeads was added per 10 8 cells, 100 μl of LFc-R blocker was added per 10 8 cells, and after thoroughly mixing, cd34+ cells were sorted by magnetic column (MACS Separation Columns) by incubation at 4 ℃ for 30min in the absence of light. CD34+ cells were cultured in 6-well plates in StemSpan TM SFEM Il (cat# 9605).
(2) Placental polypeptide, lercanidipine, and harmine-depleted induced pluripotent stem cells
CD34+ cells were cultured for 9 days, and subjected to nuclear transfection (i.e., day 0 transfection) using an Amaxa nuclear transfecter, non-integrated plasmid Epi5 TM Episomal iPSC Reprogramming Kit (cat# A15960), plasmid transfection procedure T-016, transfected cells (2X 10 6 cells) were seeded into 12-well plates, transferred 24h later to 6-well plates pre-plated with CELLADHERE TM Laminin-521 (cat# 200-0117), each well was added with 2mL of ReproTeSR TM reprogramming complete medium (cat# 05926) with a mixture of placental polypeptide, lercanidipine and harmine added, and the three were 2:5:5 (cat# ReproTeSR TM reprogramming complete medium) per day, and clones were picked up for subculture on day 15 after electric transfer.
(3) Passage and expansion of induced pluripotent stem cells
Cells were observed for confluence, and passaged at 90%. 1mL ReLeSR TM was added and incubated at 37℃for 7min. The detached cell aggregates were transferred to a 15mL centrifuge tube with a 5mL pipette, the size of the cell aggregates being 50-200 μm, which is the aggregate size suitable for plating. Aggregates were plated into culture wells coated and containing mTeSR TM Plus. When the colony density was 500, one colony contained about 500 single cells, and the culture was plated in 10 cells at a 1:10 ratio (i.e., 1 culture well cell aggregates were plated into 10 new culture wells, respectively), and the well plate was placed in a 37℃incubator. Passaging was performed every 7 days. Cultures were assessed daily with mTESR TM Plus and growth status monitored until the next passage, passing 8-10 passages until the cell line was essentially stable.
Example 3
Preparing induced pluripotent stem cells.
(1) Isolated culture of human CD34+ cells
Human peripheral blood mononuclear cells (human cordbloodmononuclear cells, MNC) were isolated by Ficoll-Paque Premium (p=1.077) density gradient centrifugation. After MNC was obtained, cd34+ cells were sorted using a Human CD34MicroBead kit, 100 μl of cd34+ Microbeads was added per 10 8 cells, 100 μl of LFc-R blocker was added per 10 8 cells, and after thoroughly mixing, cd34+ cells were sorted by magnetic column (MACS Separation Columns) by incubation at 4 ℃ for 30min in the absence of light. CD34+ cells were cultured in 6-well plates in StemSpan TM SFEM Il (cat# 9605).
(2) Placental polypeptide, lercanidipine, and harmine-depleted induced pluripotent stem cells
CD34+ cells were cultured for 9 days, subjected to nuclear transfection (i.e., day 0 of transfection) using an Amaxa nuclear transfecter, non-integrated plasmid Epi5 TM Episomal iPSC Reprogramming Kit (cat# A15960), plasmid transfection procedure T-016, and transfected cells (2X 10 6 cells) were seeded into 12-well plates, cultured for 24 hours, transferred to 6-well plates pre-plated with CELLADHERE TM Laminin-521 (cat# 200-0117), each well was added with ReproTeSR TM mL of each 2-mL complete medium (cat# 05926), and simultaneously added with a mixture of placental polypeptide, lercanidipine, and harmine at a ratio of 2:1:1. Clones were picked for subculture on day 20 after electrotransformation using ReproTeSR TM reprogramming complete medium with daily medium changes.
(3) Passage and expansion of induced pluripotent stem cells
Cells were observed for confluence, and passaged at 90%. 1mL ReLeSR TM was added and incubated at 37℃for 7min. The detached cell aggregates were transferred to a 15mL centrifuge tube with a 5mL pipette, the size of the cell aggregates being 50-200 μm, which is the aggregate size suitable for plating. Aggregates were plated into culture wells coated and containing mTeSR TM Plus. When the colony density was 500, one colony contained approximately 500 cells, and the culture was plated in 100 cells at a 1:100 ratio (i.e., 1 culture well cell aggregates were plated into 100 new culture wells, respectively), and the well plate was placed in a 37℃incubator. Passaging was performed every 7 days. Cultures were assessed daily with mTESR TM Plus and growth status monitored until the next passage, passing 8-10 passages until the cell line was essentially stable.
Comparative example 1
The only difference from example 1 is that the reprogramming complete medium does not contain placental polypeptide, which is divided in parts by weight into the weight of lercanidipine and harmine.
Comparative example 2
The only difference from example 1 is that lercanidipine was not included in the reprogramming complete medium and its parts by weight were proportioned to the weight of placental polypeptide and harmine.
Comparative example 3
The only difference from example 1 is that the reprogramming complete medium does not contain harmine, which is divided in parts by weight into the weight of placental polypeptide and lercanidipine.
Comparative example 4
The only difference from example 1 is that the reprogramming complete medium does not contain placental polypeptide and lercanidipine, which are proportioned to the weight of harmine.
Comparative example 5
The only difference from example 1 is that the reprogramming complete medium does not contain placental polypeptide and harmine, which parts by weight are proportioned to the weight of lercanidipine.
Comparative example 6
The only difference from example 1 is that the reprogramming complete medium does not contain placental polypeptide, lercanidipine, and harmine.
Comparative example 7
The only difference from example 1 is that the mass ratio of placenta polypeptide, lercanidipine and harmine is 8:1:1, respectively.
Comparative example 8
The only difference from example 1 is that the mass ratio of placenta polypeptide, lercanidipine and harmine is 1:6:6.
Test example 1
Karyotyping of induced pluripotent stem cells.
Karyotyping by chromosomal karyotyping: the P8-generation cells prepared in examples 1 to 3 and comparative examples 1 to 8 were transferred to a 6-well plate plated with Laminin-521, cultured for 2 days, and treated with 0.25g/mL of colchicine for 4 hours. Each cell line was digested with 0.05% pancreatin 500. Mu.L, neutralized by adding mTeSRTMPlus, centrifuged at 200 Xg for 5min, the supernatant was discarded, the cells harvested, and 1mL of hypotonic solution (0.4% sodium citrate and 0.4% KCl were 1:1) was added at 37℃for 5min. Adding 350 μl of the fixing solution (volume ratio of methanol to glacial acetic acid is 3:1), and centrifuging at 200×g for 5min. Removing the supernatant, adding a fixing solution (the volume ratio of methanol to glacial acetic acid is 3:1), and fixing for 40min by 4 mL. Centrifuging at 200 Xg for 5min, removing supernatant, adding 2mL of fixing solution (volume ratio of methanol to glacial acetic acid is 3:1), and fixing for 20min. Centrifuging at 200 Xg for 5min, removing supernatant, adding fresh fixing solution (methanol to glacial acetic acid volume ratio of 3:1) 200 μl, dripping into 3 sheets, air drying, and placing into 65 deg.C oven overnight. On day 2, the slides were placed in 0.25% pancreatin for 30s in a 37℃water bath for 30min. 1 drop of Giemsa stock solution is added to a glass slide, 15s is carried out, 2 drops of phosphoric acid buffer solution is added and uniformly blown, dyeing is carried out for 10min at 25 ℃, the glass slide is lightly washed by running water, and the glass slide is dried at 25 ℃. After Gemisa staining, 30 metaphase cells were randomly selected for observation and karyotyping accuracy statistics. The results are shown in Table 1. The karyotype of the induced pluripotent stem cells is 46XY type.
TABLE 1
As can be seen from table 1: the number of chromosomes of the induced pluripotent stem cells prepared in the method of the invention example 1-3 is 80% -85%, and the karyotype is the same as the normal human karyotype 46 (46, XY), which indicates that the reprogrammed induced pluripotent stem cells are normal. Comparative example 1-comparative example 6 the proportion of 46 induced pluripotent stem cell chromosomes is 70% -78%, the karyotype is normal, it is shown that placenta active polypeptide, lercanidipine and B have synergistic effect in improving the correct function of induced pluripotent stem cell karyotype. Comparative example 7-comparative example 8 the ratio of 46 chromosomes of induced pluripotent stem cells was 75% and 76%, the karyotype was normal, and it was demonstrated that the ratio of placenta-active polypeptide, lercanidipine and harmine was precisely controlled, and the ratio of normal karyotype of induced pluripotent stem cells could be improved.
Test example 2
Alkaline phosphatase detection: the P10 generation cells prepared in examples 1 to 3 and comparative examples 1 to 8 were fixed with 4% paraformaldehyde at 25℃for 5min, the paraformaldehyde fixing solution was discarded, TBST buffer was washed 2 times, AP buffer was added, equilibrated at 25℃for 6min, BCIP/NBT developing solution was added in the dark for 25min, photographs were scanned, the number of positive clones was counted, and reprogramming efficiency was calculated. re-Cheng Xiaolv (%) = (number of positive clones/number of cells in electrotransformation) ×100%. The results are shown in Table 2.
TABLE 2
| Group of | Number of positive clones | Cell number (10 5) | 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％ |
From the results, it can be seen that: the cloning efficiency of the induced pluripotent stem cells prepared in the method of the invention example 1-3 is above 0.45%. The induced pluripotent stem cells prepared by the method have high efficiency. Comparative example 1-comparative example 6 the cloning efficiency of the induced pluripotent stem cells was 0.4%, demonstrating that placenta-active polypeptide, lercanidipine and harmine have synergistic effects in improving the cloning efficiency of the induced pluripotent stem cell karyotype. Comparative example 7-comparative example 8 the cloning efficiency of the induced pluripotent stem cells obtained by the preparation of comparative example 8 was 0.4%, which indicates that the induction efficiency of cd34+ hematopoietic stem cells induced pluripotent stem cells can be improved by precisely controlling the ratio of placenta-active polypeptide, lercanidipine and harmine.
Test example 3
And (5) flow phenotype detection.
(1) Digestion: the 10 th generation cell culture solutions prepared in examples 1 to 3 and comparative examples 1 to 8 were discarded, digested with 0.25% Trypsin, terminated by adding 2 volumes of the cell basal culture solution after the cell digestion was completed, the digested cells were collected in a centrifuge tube, centrifuged at 25℃for 1000r/min,5min, and the supernatant was discarded.
(2) The detection of the flow phenotype was performed according to the instructions of the pluripotent stem cell flow detection kit (company R & D Systems, cat# FMC 001).
(3) And (5) detecting on the machine, and analyzing the cell subpopulations by using a flow cytometer. The control tube is measured first, the voltage is regulated to determine the negative area, and then the experimental tube is measured. Cells that bind to the fluorescently labeled antibody will fluoresce accordingly, thereby sorting out cells in the positive interval. The results of the flow phenotype test are shown in Table 3.
TABLE 3 Table 3
From the results, it can be seen that: the expression ratio of the induced pluripotent stem cells prepared in the method embodiment 1-3 of the invention is more than 90% of the expression ratio of the induced pluripotent stem cells OCT4 and SOX2, the expression ratio of the SSEA-4 is more than 80%, and the expression ratio of the differentiation marker gene SSEA-1 is less than 1%. The induced pluripotent stem cells prepared by the method of the invention are in accordance with the standard. Comparative example 1-comparative example 6 the results demonstrate that placenta-active polypeptide, lercanidipine and harmine have synergistic efficacy in enhancing the induced pluripotent stem cell flow phenotype. Comparative example 7-comparative example 8 the results demonstrate that the precise control of the ratio of placenta-active polypeptide, lercanidipine and harmine can provide a flow phenotype of induced pluripotent stem cells, increasing the ratio of induced pluripotent stem cells.
In summary, the invention uses non-integrated plasmid to reprogram human-derived CD34+ cells, successfully induces and forms plasmid non-integrated induced pluripotent stem cells, and the induced pluripotent stem cells have no exogenous genes and no virus insertion, avoid the danger of gene integration, have high efficiency and safety, have simplicity and repeatability, add placenta-active polypeptide, lercanidipine and harmine in the preparation process of the induced pluripotent stem cells, accurately control the addition proportion of placenta-active polypeptide, lercanidipine and harmine, and greatly improve the cloning formation rate and the accuracy of cell nucleus types of the induced pluripotent stem cells of CD34+ derived from human peripheral blood.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. A method of preparing an induced pluripotent stem cell, comprising:
The CD34+ cells were subjected to nuclear transfection using a non-integrative plasmid, and after transfection, were cultured and passaged in a reprogramming complete medium containing placental polypeptide, lercanidipine, and harmine.
2. The method of claim 1, wherein the mass ratio of placental polypeptide, lercanidipine, and harmine is (2-10): 5:5.
3. The method of claim 1 or 2, wherein the source of mononuclear cells comprises any one of human, rat, mouse, bovine, or porcine.
4. The method of any one of claims 1-3, wherein the nuclear transfection of cd34+ cells with a non-integrative plasmid comprises introducing a reprogramming gene into cd34+ hematopoietic stem cells with a non-integrative attachment vector.
5. The method of any one of claims 1-4, wherein the reprogramming genes comprise any one or a combination of at least two of OCT4, SOX2, NANOG, or SSEA-1.
6. The method of any one of claims 1-5, wherein the non-integrated, adherent vector comprises a Episomal plasmid vector.
7. The method of any one of claims 1-6, wherein the culturing is for a period of 20-30 days.
8. The method of any one of claims 1-7, wherein said passaging is communicated when cell confluence reaches 80% -90%, and wherein the passage is 8-10 passages.
9. An induced pluripotent stem cell prepared by the method for preparing an induced pluripotent stem cell according to any one of claims 1 to 8.
10. A pharmaceutical composition comprising the induced pluripotent stem cell of claim 9.
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