Preparation method of induced pluripotent stem cells
Through a multi-stage induction culture method, skin fibroblasts are reprogrammed into iPS cells, solving the problems of low iPS cell preparation efficiency and carcinogenic risk, achieving efficient and safe cell reprogramming, and providing a new therapeutic strategy.
Patent Information
- Application Number
- CN202510283627.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-04-22
AI Technical Summary
The preparation of induced pluripotent stem cell (iPS) cells is inefficient and has a risk of carcinogenicity, making it difficult to apply to disease treatment.
Through a multi-stage induction culture method, skin fibroblasts were reprogrammed into iPS cells, and small-molecular compounds were used to replace retroviral and lentiviral vectors, inducing cells to express pluripotency markers and form teratomas in trigerm tissue.
The efficient reprogramming of skin fibroblasts into iPS cells is achieved, reducing the risk of carcinogenicity and providing a safer and more efficient therapeutic reprogramming strategy.
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Figure CN119859607A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of cell biotechnology, and in particular to a method for preparing induced pluripotent stem cells. Background Art
[0002] Stem cells are divided into adult stem cells and embryonic stem cells (ES). ES cells are diploid cells isolated from the inner cell mass of the blastocyst, which have the potential to develop and differentiate into almost all tissue cell types in the body. However, there has been fierce ethical debate in the study of ES cells. In order to avoid ethical debates, foreign scholars have induced somatic cell gene reprogramming through somatic cell in vitro gene transfection technology to obtain induced pluripotent stem (iPS) cell lines. Nuclear reprogramming refers to the process in which mature cells are reversed from a differentiated state to an undifferentiated state. Since 2008, the research on iPS cells in mice and humans has made great progress, and has been improved in the selection of somatic cells, the combination and number of transcription factors, viral vectors, screening conditions and small molecule compounds. It has also gradually deepened into the treatment of diseases, laying the foundation for the treatment of human monogenic genetic diseases in theory and practice. With the establishment of more and more research models, more and more somatic cells have been successfully induced into pluripotent stem cells, as well as the discovery of virus-free integration technology and the successful knockout of harmful genes, induced pluripotent stem cells have developed in a more systematic, safer and more efficient direction.
[0003] With the success of inducing iPS cells to differentiate into dopamine neurons, cells that can secrete insulin, cardiac parenchymal cells, retinal progenitor cells and neural progenitor cells, more and more somatic cells have been successfully induced to differentiate, providing the possibility of cure for patients with Parkinson's syndrome, diabetes, cardiovascular disease, retinopathy and other diseases.
[0004] However, iPS cells still face two major problems: First, the efficiency of iPS cell preparation is very low, generally only 0.05%, so it is difficult to apply. Second, iPS cells have great risks, because the vectors generally use retroviruses and lentiviruses, and the oct, c-Myc, klf4, and sox2 genes can all be considered proto-oncogenes, so iPS cells are extremely carcinogenic. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing induced pluripotent stem cells, which can successfully induce skin fibroblasts into induced pluripotent stem cells, providing a new means for the treatment of related diseases.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing induced pluripotent stem cells, comprising the following steps: S1, inoculating fibroblasts into a first culture medium for a first induction culture; S2, replacing the first culture medium with the second culture medium, and performing a second induction culture; S3, replacing the second culture medium with the third culture medium, performing the third induction culture, and obtaining cell colonies with a typical ES colony dense morphology; S4, digesting the cell colonies to obtain dispersed cells, inoculating them into the fourth culture medium, performing subculture, and obtaining induced pluripotent stem cells; The third culture medium contains CHIR99021, PD0325901, SB590885, IWP2 and Y27632.
[0007] Preferably, the fibroblasts are P2 fibroblasts.
[0008] Preferably, the first culture medium contains CHIR99021, 616452, TTNPB, Y27632, ABT869 and SAG.
[0009] Preferably, the final concentration of CHIR99021 in the first culture medium is 0.4-0.6 μM; The final concentration of 616452 in the first culture medium is 8-12 μM; The final concentration of TTNPB in the first culture medium is 18-22 μM; The final concentration of Y27632 in the first culture medium is 0.05-0.15 μM; The final concentration of ABT869 in the first culture medium is 1-3 μM; The final concentration of SAG in the first culture medium is 8-12 μM.
[0010] Preferably, the basal culture medium in the first culture medium is liquid No. 0 of iPSC culture medium.
[0011] Preferably, in S1, the inoculation is performed using a 12-well plate; During the inoculation, the peripheral holes are filled with physiological saline, and the two middle holes are inoculated with the fibroblasts; The inoculation amount of the fibroblasts is 0.5-2×104 / ml / well; The first induction culture time is 6 to 10 days.
[0012] Preferably, the second culture medium contains JNKIN8, 5-azacytidine and tranylcypromine.
[0013] Preferably, the final concentration of JNKIN8 in the second culture medium is 1.5-2.5 μM; The final concentration of 5-azacytidine in the second culture medium is 0.5-1.5 μM; The final concentration of tranylcypromine in the second culture medium is 3-8 μM.
[0014] Preferably, the basal culture medium in the second culture medium is liquid I of the iPSC culture medium; The second induction culture time is 6 to 10 days.
[0015] Preferably, the final concentration of CHIR99021 in the third culture medium is 0.3-0.8 μM; The final concentration of PD0325901 in the third culture medium is 1.5-2.5 μM; The final concentration of SB590885 in the third culture medium is 0.05-0.15 μM; The final concentration of IWP2 in the third culture medium is 0.3-0.8 μM; The final concentration of Y27632 in the third culture medium is 0.5-1.5 μM; And / or, the basal culture medium in the third culture medium is liquid II of the iPSC culture medium; And / or, the third induction culture time is 10 to 15 days; And / or, the fourth culture medium is liquid III of iPSC culture medium; And / or, the digestion uses TrypLETM Express enzyme.
[0016] Beneficial effects of the present invention: The present invention provides a safer and more efficient technical means to successfully achieve the reprogramming of skin fibroblasts into iPS. The present invention uses small molecule compounds to replace retroviral and lentiviral vectors, and the induced pluripotent stem cells have the characteristics of pluripotency, including the expression of OCT4, SOX2, Nanong pluripotency markers and the ability to form teratomas of three germ layer tissues. The present invention successfully utilizes the characteristics of small molecules not integrating with the genome, being highly controllable, easy to optimize, standardize and manufacture, so that the resulting chemical reprogramming method is expected to develop therapeutic reprogramming strategies in a highly controllable and easy to standardize manner. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 A schematic diagram of the preparation method provided by the present invention; Figure 2 is a graph of gene expression; Figure 3 Diagram showing stage 1 status for immunofluorescence and reverse transcription quantitative PCR (RT-qPCR) analysis; Figure 4Diagram showing stage 2 status for immunofluorescence and reverse transcription quantitative PCR (RT-qPCR) analysis; Figure 5 Diagram showing stage 3 status for immunofluorescence and reverse transcription quantitative PCR (RT-qPCR) analysis; Figure 6 Figure 4 shows the situation of stage 4 for immunofluorescence and reverse transcription quantitative PCR (RT-qPCR) analysis; Figure 7 This is the result of microscopic observation of hES cell morphology; Figure 8 This is the electron microscopic observation result of the teratoma of the three germ layer tissues; Fig. 9 Schematic diagram of vaccination method; Fig.10 This is a morphological diagram of fibroblasts; Fig.11 The cell morphology pictures after fibroblast inoculation, the left picture is the 3rd day after inoculation, the middle picture is the 6th day after inoculation, and the right picture is the 9th day after inoculation; Fig.12 These are the culture status diagrams on days 4, 8 and 12 of the third stage. DETAILED DESCRIPTION
[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0019] Example 1 The preparation method of the present invention comprises: (1) obtaining a gene program similar to regeneration; (2) activating the gene program; and (3) establishing a pluripotency grid. Through the above steps, fibroblast-induced pluripotent stem cells can be effectively prepared, and can express the characteristics of pluripotent stem cells, including expressing OCT4, SOX2, and Nanong pluripotency markers. The overall preparation process is as follows Figure 1 .
[0020] To induce fibroblast dedifferentiation in step (1), we used small molecules to disrupt fibroblast characteristics, promote cell proliferation, and reactivate dedifferentiation-related genes. The small molecule compound combination: CHIR99021, 616452, TTNPB, Y27632, ABT869, and SAG. This treatment upregulated epithelial cell-related genes (KRT8, KRT18, and KRT19) and downregulated a group of fibroblast marker genes ( Figure 2 ), indicating a loss of fibroblastic identity. Immunofluorescence and reverse transcription quantitative PCR (RT-qPCR) analysis showed that the expression of LIN28A, an important gene regulating dedifferentiation and regeneration in different species, was decreased in the cultures ( Figure 3 ).
[0021] For activation of the gene program in step (2), JNKIN8, 5-azacytidine, and tranylcypromine were added to induce a hypomethylated and proliferative cell state. After treatment, SALL4, a key transcription factor that regulates dedifferentiation of embryonic cells, was activated and co-expressed with LIN28A ( Figure 4 ), indicating the characteristics of cell dedifferentiation.
[0022] For step (3) pluripotency gene network, CHIR99021, PD0325901, SB590885, IWP2 and Y27632 were added to induce dense colonies co-expressing OCT4, SOX2 and NANOG ( Figure 5 and Figure 6 After transfer into human embryonic stem (hES) cell culture medium, these colonies showed typical hES cell morphology ( Figure 7 ), teratomas that can form three germ layer tissues can ( Figure 8 ).
[0023] Sources of materials used in the examples: Human fibroblasts were isolated and cultured to passage P2.
[0024] Preparation of the culture medium for the four stages. The iPSC culture medium used in the subsequent implementation cases is the commercial production culture medium of Saijiyuan Biotechnology (name: ApolloMSC; item number: SJY-ApolloMSC-001), including 50ml of solution 0, 100ml of solution I, 100ml of solution II and 100ml of solution III. The culture medium is stored at -20℃ and thawed at room temperature or in a 4℃ refrigerator overnight before use.
[0025] TrypLE from Gibco TM Express, can be used directly.
[0026] The small molecule compounds used in the present invention include: CHIR99021, 616452, TTNPB, Y27632, ABT869, SAG, JNKIN8, 5-azacytidine, tranylcypromine, PD0325901, SB590885, IWP2 and Y27632 (all purchased from Beyotime).
[0027] in: 1 ml of DMSO was added to every 5.02 mg of CHIR99021 to prepare a 10 mM solution.
[0028] 616452 was prepared into a 10 mM solution by adding 1 ml of DMSO for every 2.87 mg.
[0029] TTNPB was prepared into a 10 mM solution by adding 1 ml of DMSO for every 3.48 mg of TTNPB.
[0030] Y27632 was prepared into a 10 mM solution by adding 1 ml of DMSO for every 3.20 mg of Y27632.
[0031] ABT869 was prepared into a 10 mM solution by adding 1 ml of DMSO for every 3.75 mg of ABT869.
[0032] For every 5.27 mg of SAG, 1 ml of DMSO was added to prepare a 10 mM solution.
[0033] 1 ml of DMSO was added for every 5.08 mg of JNKIN8 to prepare a 10 mM solution.
[0034] 1 ml of DMSO was added for every 2.44 mg of 5-azacytidine to prepare a 10 mM solution.
[0035] For every 1.82 mg of tranylcypromine, add 1 ml of DMSO to prepare a 10 mM solution.
[0036] 1 ml of DMSO was added to every 4.82 mg of PD0325901 to prepare a 10 mM solution.
[0037] SB590885 was prepared into a 10 mM solution by adding 1 ml of DMSO for every 4.31 mg of SB590885.
[0038] 1 ml of DMSO was added to every 4.67 mg of IWP-2 to prepare a 10 mM solution.
[0039] Y27632 was prepared into a 10 mM solution by adding 1 ml of DMSO for every 3.20 mg of Y27632.
[0040] CHIR99021, 616452, TTNPB, Y27632, ABT869 and SAG were added to solution 0 with final concentrations of 0.5 μM, 10 μM, 20 μM, 0.1 μM, 2 μM and 10 μM, respectively.
[0041] JNKIN8, 5-azacytidine and tranylcypromine were added to solution I to a final concentration of 2 μM, 1 μM and 5 μM, respectively.
[0042] CHIR99021, PD0325901, SB590885, IWP2 and Y27632 were added to solution II with final concentrations of 0.5 μM, 2 μM, 0.1 μM, 0.5 μM and 1 μM, respectively.
[0043] To cultivate: Collect P2 fibroblasts, resuspend the cell suspension with physiological saline, mix well, take 20 μL of cell suspension and add it to 20 μL of trypan blue, take out 20 μL of the mixture for counting. According to the counting results, calculate 2.0*10 8 The volume of cell suspension was calculated and centrifuged at 300g for 6 min. After centrifugation, the supernatant was removed and the cell pellet was resuspended in 2 ml of No. 0 solution and inoculated into a 12-well plate. The outer wells of the 12-well plate were filled with 1.5 ml of saline per well. The fibroblast suspension was inoculated in the middle two wells, 1*10 4 cells / ml / well. Fig. 9 Inoculate as shown. Change the medium every 3 days and culture for 8 days. This stage is stage 1.
[0044] After 8 days of induction, remove solution 0 and add solution 1, 1 ml / well. Change the solution every 3 days and culture for 8 days. This stage is stage 2.
[0045] After 8 days of induction, remove solution I and add solution II at 1 ml / well. Change the solution every 3 days and culture for 12 days until the cells grow into 5-8 colonies with a typical dense morphology of ES colonies. This stage is stage 3.
[0046] After 12 days of induction, remove solution II, rinse the wells twice with saline, and add TrypLE TM Express enzyme, 1ml / well, digest for 5min, add 2ml / well of physiological saline to terminate digestion, blow the cells into single cells with a sterile pipette, take 20μL of cell suspension and add it to 20μL of trypan blue, take out 20μL of the mixture for counting. Centrifuge at 300g, 6min, remove the supernatant, and centrifuge at 5*10 4 / well were inoculated into 6-well plates and cultured with liquid culture medium No. III to start P1 generation. This stage is stage 4.
[0047] When the cell confluence reaches 90%, the cells are passaged to the next generation. It is best to keep the passage time stable at 3-4 days.
[0048] Figure 10-12 The changes in cell morphology during the induction of fibroblasts into iPS cells are summarized. Fig.10 The morphology of fibroblasts. Fig.11 On the third day after the fibroblasts were inoculated, it was observed that the cells changed from "fibroblast-like" to "endothelial-like", and the cell fusion degree continued to increase. Fig.11 On the 3rd to 9th day after inoculation, it can be observed that the number and deformation of cells gradually increase, the deformation of cells is greater than 90%, and the cell fusion degree reaches 90%. Fig.12On the 4th, 8th and 12th days of the third stage, multilayer cell clones were observed. As the culture continued, the number and range of multilayer clones gradually increased.
[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing induced pluripotent stem cells, characterized in that: The following steps are involved: S1, inoculating fibroblasts into a first culture medium for a first induction culture; S2, replacing the first culture medium with a second culture medium, and performing a second induction culture; S3, replacing the second culture medium with the third culture medium, performing the third induction culture, and obtaining cell colonies with a typical ES colony dense morphology; S4, digesting the cell colonies to obtain dispersed cells, inoculating them into the fourth culture medium, performing subculture, and obtaining induced pluripotent stem cells; The third culture medium contains CHIR99021, PD0325901, SB590885, IWP2 and Y27632.
2. The preparation method according to claim 1, characterized in that: The fibroblasts are P2 generation fibroblasts.
3. The preparation method according to claim 1, characterized in that: The first culture medium contains CHIR99021, 616452, TTNPB, Y27632, ABT869 and SAG.
4. The preparation method according to claim 3, characterized in that: The final concentration of CHIR99021 in the first culture medium is 0.4-0.6 μM; The final concentration of 616452 in the first culture medium is 8-12 μM; The final concentration of TTNPB in the first culture medium is 18-22 μM; The final concentration of Y27632 in the first culture medium is 0.05-0.15 μM; The final concentration of ABT869 in the first culture medium is 1-3 μM; The final concentration of SAG in the first culture medium is 8-12 μM.
5. The preparation method according to claim 4, characterized in that: The basal culture medium in the first culture medium is liquid No. 0 of the iPSC culture medium.
6. The preparation method according to claim 1, characterized in that: In S1, the inoculation was performed using a 12-well plate; During the inoculation, the peripheral holes are filled with physiological saline, and the two middle holes are inoculated with the fibroblasts; The inoculation amount of the fibroblasts is 0.5~2×10 4 pcs / ml / well; The first induction culture time is 6 to 10 days.
7. The preparation method according to claim 1, characterized in that: The second culture medium contains JNKIN8, 5-azacytidine and tranylcypromine.
8. The preparation method according to claim 7, characterized in that: The final concentration of JNKIN8 in the second culture medium is 1.5-2.5 μM; The final concentration of 5-azacytidine in the second culture medium is 0.5-1.5 μM; The final concentration of tranylcypromine in the second culture medium is 3-8 μM.
9. The preparation method according to claim 8, characterized in that: The basal culture medium in the second culture medium is liquid I of the iPSC culture medium; The second induction culture time is 6 to 10 days.
10. The preparation method according to claim 1, characterized in that: The final concentration of CHIR99021 in the third culture medium is 0.3-0.8 μM; The final concentration of PD0325901 in the third culture medium is 1.5-2.5 μM; The final concentration of SB590885 in the third culture medium is 0.05-0.15 μM; The final concentration of IWP2 in the third culture medium is 0.3-0.8 μM; The final concentration of Y27632 in the third culture medium is 0.5-1.5 μM; And / or, the basal culture medium in the third culture medium is liquid II of the iPSC culture medium; And / or, the third induction culture time is 10 to 15 days; And / or, the fourth culture medium is liquid III of iPSC culture medium; And / or, the digestion is carried out using TrypLE TM Express enzyme.