Method for reprogramming human somatic cells into induced pluripotent stem cells

Abstract

A simpler and more efficient method for reprogramming human somatic cells into induced pluripotent stem cells. According to the method, three transcription factors of OCT4, SOX2, and LIN28 are simultaneously transferred into somatic cells, and then reprogramming is performed under the co-induction of four small molecules of Peficitinib, VTP50469, SB203580, and FPFT-2216, wherein the peripheral blood reprogramming efficiency thereof can reach 0.02%, the induction period is only 14-16 days, and the system is stable.

Description

Methods for reprogramming human cells into induced pluripotent stem cells Technical Field

[0001] The present invention relates to the technical field of pluripotent stem cell reprogramming, and in particular to a simpler and more efficient method for reprogramming human cells into induced pluripotent stem cells. Background Art

[0002] Since Shinya Yamanaka's team induced mouse fibroblasts into pluripotent stem cells in 2006, pluripotent stem cell reprogramming technology has experienced rapid development, and human somatic cell reprogramming has also been successfully achieved step by step. For example, Yu Junying's team reprogrammed peripheral blood into induced pluripotent stem cells by introducing six transcription factors (OCT4, SOX2, LIN28, NANOG, C-MYC, and KLF4). Miguel Esteban and Pei Duanqing's teams successfully reprogrammed kidney epithelial cells into induced pluripotent stem cells by introducing three transcription factors (OCT4, SOX2, KLF4) and micro RNA302 / 367 into urine cells. In 2022, Deng Hongkui's team successfully reprogrammed human fibroblasts into pluripotent stem cells using complete small-molecule reprogramming, but the induction time was too long.

[0003] During the process of somatic cell reprogramming into induced pluripotent stem cells, the fate of the cells undergoes drastic changes, and characteristic genes also undergo transformation. Scientists now have a general understanding of the genes that are completely suppressed and completely activated after somatic cells are transformed into stem cells. By adding small molecules that promote the relevant pathways of the genes to be activated and small molecules that inhibit the relevant pathways of the genes originally to be silenced, we can achieve efficient and stable reprogramming solutions with a small amount of transcription factors. However, research on reprogramming with a small number of transcription factors is currently progressing slowly, and the few reported schemes still suffer from difficulties in reproducibility and unstable efficiency. For example, the invention patent with publication number CN114645023B, "System and method for reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells," has a reprogramming efficiency of only about 0.006%. Moreover, the number of clones varies greatly in repeated experiments, and the efficiency fluctuates between 0.002-0.006%, which is very unstable. Therefore, in order to gain a deeper understanding of the role of different transcription factors in the reprogramming of somatic cells to embryonic stem cells and enrich the schemes for the conversion of somatic cells to embryonic stem cells, it is necessary to find a reprogramming scheme with high efficiency and high reproducibility that is more suitable for peripheral blood cells, a cell type that is difficult to transfect.

[0004] Summary of the Invention

[0005] Based on this, one of the objects of the present invention is to provide a method for reprogramming human cells into induced pluripotent stem cells, comprising the following steps: only three reprogramming induction factors, OCT4, SOX2, and LIN28, are transferred into somatic cells, and then cultured in the presence of chemical inducers to obtain induced pluripotent stem cells, wherein the chemical inducers are composed of Peficitinib, VTP50469, SB203580, and FPFT-2216, the concentration of Peficitinib is 0.5-1.5 μM, the concentration of VTP50469 is 0.1-1 μM, the concentration of SB203580 is 1-3 μM, and the concentration of FPFT-2216 is 1-5 μM.

[0006] As a preferred embodiment, the three reprogramming inducing factors, OCT4, SOX2 and LIN28, are introduced into somatic cells in the form of nucleic acids or protein products.

[0007] As a preferred embodiment, the three reprogramming inducing factors OCT4, SOX2 and LIN28 are introduced into somatic cells in the form of their DNA or mRNA.

[0008] As a preferred embodiment, the three reprogramming inducing factors OCT4, SOX2 and LIN28 are transferred into somatic cells in forms including but not limited to Sendai virus transfection system, mRNA transfection system and episomal plasmid transfection system.

[0009] As a preferred embodiment, the somatic cells are hematopoietic precursor cells derived from peripheral blood.

[0010] As a preferred embodiment, a method for reprogramming human cells into induced pluripotent stem cells comprises the following steps: transferring the three transcription factors OCT4, SOX2, and LIN28 into somatic cells, inducing them using an induction medium containing the chemical inducer described in claim 1, and finally culturing them using a pluripotent stem cell medium until the induced pluripotent stem cell clones are fully mature, and then randomly picking clones for expansion; wherein the induction medium is a pluripotent stem cell medium supplemented with 2-4 μM chir99021, 80-120 ng / ml FGF2, 200-300 μM NaB, 1-3 μM SB203580, 0.5-1.5 μM peficitinib, 0.05-0.2 μM VTP50469, 1-5 μM FPFT-2216, 1-3% B27 by volume, and 1% N2 by volume; the pluripotent stem cell medium is mTeSR1 medium.

[0011] Specifically, the three transcription factors OCT4, SOX2, and LIN28 are transferred into somatic cells by electrofection of an episomal plasmid. The episomal plasmid is a modified pCPE4 vector carrying the three transcription factors, wherein the two transcription factors OCT4 and SOX2 are constructed on the same pCPE4 vector.

[0012] Specifically, after electroporation, peripheral blood expansion medium is immediately added, and the cells are seeded in a well plate coated with a basement membrane matrix and cultured for 46 to 50 hours; induction medium is added, and culture is continued for 46 to 50 hours. Subsequently, new induction medium is added using an incomplete medium exchange method to continue induction for 46 to 50 hours. The operation is repeated 1 to 3 times, and the old medium is completely discarded. Pluripotent stem cell medium is added for culture, and the pluripotent stem cell culture medium is replaced every day until the induced pluripotent stem cell clones are fully mature, and clones can be picked and expanded at any time.

[0013] Specifically, the peripheral blood expansion medium is a serum-free medium supplemented with SCF 80-120 ng / mL, FLT-3 80-120 ng / mL, IL-3 10-30 ng / mL, IL-6 10-30 ng / mL, and TPO 80-120 ng / mL.

[0014] The present invention reprograms human peripheral blood cells into induced pluripotent stem cells under the action of three transcription factors and four small molecule inhibitors. The reprogramming efficiency can reach 0.02%, which is about 3.3 times higher than the existing technology (patent number CN114645023B). The induction period is only 14 to 16 days, and the system is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows the microscopic observation during the induction process of treatment group 1;

[0016] FIG2 is a statistical comparison of the number of clones obtained in different induction treatment groups in Example 1;

[0017] FIG3 shows the expression of pluripotent cell markers by flow cytometry for pluripotent stem cells obtained from treatment group 1;

[0018] Figure 4 shows the immunofluorescence detection results of pluripotent stem cell markers, DAPI staining fluorescence detection results, and Merge results obtained in treatment group 1;

[0019] FIG5 shows the purity analysis results of induced pluripotent stem cells obtained in treatment group 1;

[0020] FIG6 shows the results of the tumor formation experiment of induced pluripotent stem cells obtained in treatment group 1 in mice. DETAILED DESCRIPTION

[0021] Below in conjunction with specific embodiment, the present invention is described in further detail so that those skilled in the art can understand the present invention more clearly.The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention.Based on the specific embodiments in the present invention, those of ordinary skill in the art, without making creative work, all other embodiments obtained belong to protection scope of the present invention.

[0022] Unless otherwise specified, all reagents used in the examples of the present invention are commercially available. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0023] The present invention takes peripheral blood cells as an example. After they are transformed into stem cells, the signal pathways that originally characterized their blood cell fate are silenced. These signal pathways include: JAK-STAT signal pathway, AKT signal pathway, IK2F1 gene pathway, PTPN22 gene pathway and TAL1 gene pathway, etc. The present invention optimizes the reprogramming induction factors to three factors: OCT4, SOX2, and LIN28. At the same time, they are induced and cultured under the induction of four chemical small molecules: Peficitinib, VTP50469, SB203580, and FPFT-2216, thereby achieving the transformation of peripheral blood cells into induced pluripotent stem cells. In particular, for hematopoietic precursor cells derived from peripheral blood, the reprogramming efficiency can reach 0.02%, the induction period is only 14 to 16 days, and the system is more stable.

[0024] The three induction factors OCT4, SOX2, and LIN28 optimized and determined in the present invention are all reported induction factors, among which the sequence information of the OCT4 transcription factor is accession number 5460 on NCBI, the sequence information of the SOX2 transcription factor is accession number 6657 on NCBI, and the sequence information of the LIN28 transcription factor is accession number 79727 on NCBI.

[0025] The chemical inducer "Peficitinib" described in the present invention is an inhibitor of Janus kinase (JAK), purchased from MCE with the product number HY19568.

[0026] The chemical inducer "VTP50469" described in the present invention is a Menin-MLL interaction inhibitor purchased from MCE, product number: HY-114162.

[0027] The chemical inducer "SB203580" described in the present invention is a p38 MAPK inhibitor purchased from MCE, product number: HY10256.

[0028] The chemical inducer "FPFT-2216" described in the present invention is a zinc finger transcription factor IKZF1, purchased from MCE, item number: HY-145319.

[0029] The SCF described in the present invention is stem cell factor, FLT-3 is type III receptor tyrosine kinase, IL-3 is interleukin-3, IL-6 is interleukin-6, and TPO is thyroid peroxidase (TPO).

[0030] StemPro TM -34 medium was purchased from Gbico, catalog number 10640-019.

[0031] The chir99021 described in the present invention is a glycogen synthase kinase 3 (GSK-3) inhibitor, FGF2 is fibroblast growth factor 2, NaB is the sodium salt form of butyrate, and is a histone deacetylase (HDAC) inhibitor, B27 is a serum-free additive, and N-2 is an additive.

[0032] mTeSR1 culture medium was purchased from Stemcell, catalog number 85850.

[0033] The chemical inducer "Tofacitinib" in the comparative example of the present invention is a JAK3 inhibitor purchased from MCE, product number: HY-40354.

[0034] In the present invention, the three reprogramming inducing factors OCT4, SOX2 and LIN28 are introduced into somatic cells in the form of, but not limited to, nucleic acid forms, such as DNA forms, mRNA forms, or in the form of their protein products.

[0035] In the present invention, the forms of transferring the three reprogramming inducing factors OCT4, SOX2 and LIN28 into somatic cells include but are not limited to Sendai virus transfection system, mRNA transfection system and episomal plasmid transfection system.

[0036] Example 1 Comparison of reprogramming under different induction factors

[0037] Treatment groups 1 to 6 all used episomal plasmid electroporation to introduce induction factors. The only difference between the treatment groups was the addition of chemical induction small molecules to the pluripotent stem cell induction medium. The specific steps are as follows:

[0038] (1) Construction of episomal plasmids:

[0039] Construction of the PCEP4-OCT4-IRES-SOX2 plasmid: Purchase the empty vector plasmid pCEP4 from the thermo website (catalog number V04450). Download the OCT4 and SOX2 gene sequences from NCBI (accession numbers 5460 and 6657 for the OCT4 and SOX2 transcription factors, respectively). Ligate the target gene (transcription factor gene sequence) between the SnaBI and AscI restriction sites on the empty vector plasmid pCEP4 via homologous recombination.

[0040] Construction of the PCEP4-Lin28 plasmid: Purchase the empty vector plasmid pCEP4 from the thermo website (catalog number V04450). Download the Lin28 gene sequence from NCBI (accession number 79727). Ligate the target gene (Lin28 transcription factor gene sequence) into the empty vector plasmid pCEP4 between the SnaBI and AscI restriction sites via homologous recombination.

[0041] (2) Peripheral blood cell expansion: Freshly isolated peripheral blood cells (ethically qualified peripheral blood cells were purchased from Shanghai HYCELLs Company) were cultured at a rate of 5×10 6 / mL was seeded in a six-well plate and cultured in a 37°C incubator for 7 days. The peripheral blood cell expansion medium was StemPro supplemented with SCF 100ng / mL, FLT-3 100ng / mL, IL-3 20ng / mL, IL-6 20ng / mL and TPO 100ng / mL. TM -34medium.

[0042] (3) Electrotransfection: On the day before electrotransfection, 500 μL of diluted basement membrane matrix (Matrigrl) was added to the 12-well plate and placed in a carbon dioxide incubator for coating. The amplified cells were removed, washed and counted, and the counted peripheral blood mononuclear cells (PBMC) were counted at 2×10 6 / mL density was added to a 1.5mL centrifuge tube, and the centrifuge tube was loaded into a centrifuge. The centrifugal parameters were set to a centrifugal force of 200g and a time of 5 minutes. After centrifugation, the supernatant was aspirated with a pipette for later use;

[0043] According to the operating procedures of Thermo Neon electroporation instrument, buffer was added, 5 μg PCEP4-OCT4-IRES-SOX2 plasmid and 2 μg PCEP4-Lin28 plasmid were mixed with cell pellet and inserted into the electroporation instrument for electroporation. Then, peripheral blood expansion medium was immediately added and the cells were cultured at a rate of 5×10 5 The cells were seeded at a density of 100 μg / mL in a coated six-well plate and placed in a 37°C carbon dioxide incubator for 48 h.

[0044] (4) Induction: 48 hours after electroporation, add 1 mL of pluripotent stem cell induction medium directly to the original well plate, and continue to place it in a 37°C carbon dioxide incubator for 48 hours; on the fourth day after electroporation, aspirate half of the old medium in the original well plate, then add 1 mL of fresh pluripotent stem cell induction medium, and continue to place it in a 37°C carbon dioxide incubator for 48 hours; on the sixth day after electroporation, aspirate all of the old medium in the original well plate, then add 1 mL of fresh pluripotent stem cell induction medium, and continue to place it in a 37°C carbon dioxide incubator for 48 hours. 48h; on the eighth day after electroporation, all the old culture medium in the original well plate was aspirated and discarded, and then 1mL of fresh pluripotent stem cell induction medium was added, and the cells were placed in a 37℃ carbon dioxide incubator for 48h; on the tenth day after electroporation, all the old culture medium in the original well plate was aspirated and discarded, and then 1mL of fresh mTeSR1 medium was added, and the cells were placed in a 37℃ carbon dioxide incubator for culturing; mTeSR1 medium was replaced every day until the induced pluripotent stem cells were fully mature, which was approximately the fourteenth or fifteenth day after electroporation, and then clones were picked for expansion.

[0045] Among them, the pluripotent stem cell induction medium used in treatment group 1 was mTeSR1 supplemented with 3μM chir99021, 100ng / ml FGF2, 250μM NaB, 2μM SB203580, 1μM peficitinib, 1μM FPFT-2216, 0.1μM VTP50469, 2% B27 by volume, and 1% N-2 by volume.

[0046] The pluripotent stem cell induction medium used in treatment group 2 was 3 μM chir99021, 100 ng / ml FGF2, 250 μM NaB, 1 μM peficitinib, 2% B27 by volume, and 1% N-2 by volume in mTeSR1.

[0047] The pluripotent stem cell induction medium used in treatment group 3 was 3 μM chir99021, 100 ng / ml FGF2, 250 μM NaB, 2 μM Tofacitinib, 2% B27 by volume, and 1% N-2 by volume in mTeSR1.

[0048] The pluripotent stem cell induction medium used in treatment group 4 was 3 μM chir99021, 100 ng / ml FGF2, 250 μM NaB, 2 μM SB203580, 2% B27 by volume, and 1% N-2 by volume in mTeSR1.

[0049] The pluripotent stem cell induction medium used in treatment group 5 was 3 μM chir99021, 100 ng / ml FGF2, 250 μM NaB, 2 μM SB203580, 1 μM peficitinib, 1 μM FPFT-2216, 2% B27 by volume, and 1% N-2 by volume in mTeSR1.

[0050] Treatment group 6 did not add inducing small molecules.

[0051] The number of induced pluripotent stem cell clones in each treatment group was counted and compared:

[0052] Alkaline phosphatase staining experiment: Alkaline phosphatase staining kit was purchased from Yisheng Biotechnology with the catalog number 40749ES. DAY14 cell samples from treatment groups 1 to 6 were stained according to the instructions. Since induced pluripotent stem cells highly express alkaline phosphatase, purple induced pluripotent stem cell clones appeared in the well plate. Subsequently, Image J software was used to count the clones, that is, every 5.0×10 5 The results of clones after PBMC reprogramming are shown in Table 1 and Figure 2 below:

[0053] Table 1 Comparison of programming effects of different reprogramming methods

[0054] As shown in Table 1, more pluripotent stem cell clones can be obtained by reprogramming induction using treatment group 1, and the reprogramming efficiency can reach 0.02%. The system is stable and has a high reproducibility. The results of electron microscopic observation of human peripheral blood mononuclear cells (PBMCs) in treatment group 1 before, after, and 9 and 16 days after treatment are shown in Figure 1.

[0055] Example 2 Identification of Induced Pluripotent Stem Cells Obtained from Treatment Group 1

[0056] The induced pluripotent stem cells obtained from treatment group 1 were characterized and identified using different methods including flow cytometry, immunofluorescence, and quantitative PCR.

[0057] The induced pluripotent stem cells obtained from treatment group 1 were subjected to immunofluorescence detection of the following markers, OCT4 and NANOG, by immunofluorescence technology. The results are shown in Figure 3, wherein the first row shows the immunofluorescence detection results of the OCT4 marker, the DAPI staining fluorescence detection results, and the Merge results, respectively; the second row shows the immunofluorescence detection results of the NANOG marker, the DAPI staining fluorescence detection results, and the Merge results, respectively.

[0058] The obtained induced pluripotent stem cells were analyzed by flow cytometry for the following molecular markers: TRA-1-60-R and SSEA-3. The results are shown in FIG4 , demonstrating that the obtained cells have the characteristics of pluripotent stem cells.

[0059] Example 3 Purity Analysis of Induced Pluripotent Stem Cells Obtained in Treatment Group 1

[0060] According to the immunofluorescence detection and flow cytometry results in Example 2, combined with the data statistics using Image J software, it can be seen that the proportion of cells representing pluripotency markers (OCT4 / NANOG / TRA-1-60-R / SSEA3) reached more than 95%, as shown in Figure 5.

[0061] Example 4 Tumor formation experiment in mice

[0062] Take 5×10 6 The iPSC-P15 cells (i.e., 15th generation cells) obtained from treatment group 1 were resuspended in a 1:1 mixture of 50 μL DMEMF12 and 50 μL Matrigel matrix gel and placed on ice. The cell mixture was then injected subcutaneously into the axilla of the left upper limb of immunodeficient mice using an insulin injection needle. The cells were allowed to proliferate in vivo for 8-12 weeks. When obvious tumor-like protrusions appeared under the axilla, they were removed for paraffin sectioning and HE staining to observe the differentiation of the three germ layers.

[0063] As shown in Figure 6 , iPS cells can differentiate into three germ layers in vivo and form complete teratomas, demonstrating their pluripotent differentiation ability.

[0064] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for reprogramming human cells into induced pluripotent stem cells, characterized in that: The method comprises the following steps: only three reprogramming induction factors, namely OCT4, SOX2 and LIN28, are transferred into somatic cells, and then cultured in the presence of chemical inducers to obtain induced pluripotent stem cells, wherein the chemical inducers are composed of Peficitinib, VTP50469, SB203580 and FPFT-2216, the concentration of Peficitinib is 0.5 to 1.5 μM, the concentration of VTP50469 is 0.1 to 1 μM, the concentration of SB203580 is 1 to 3 μM, and the concentration of FPFT-2216 is 1 to 5 μM.

2. The method for reprogramming human cells into induced pluripotent stem cells according to claim 1, characterized in that: The three reprogramming inducing factors, OCT4, SOX2 and LIN28, are introduced into somatic cells in the form of their nucleic acids or their protein products.

3. The method for reprogramming human cells into induced pluripotent stem cells according to claim 1, characterized in that: The three reprogramming inducing factors, OCT4, SOX2 and LIN28, are introduced into somatic cells in the form of their DNA or mRNA.

4. The method for reprogramming human cells into induced pluripotent stem cells according to claim 1, characterized in that: The forms of transferring the three reprogramming inducing factors OCT4, SOX2 and LIN28 into somatic cells include but are not limited to Sendai virus transfection system, mRNA transfection system and episomal plasmid transfection system.

5. The method for reprogramming human cells into induced pluripotent stem cells according to claim 1, characterized in that: The somatic cells are hematopoietic precursor cells derived from peripheral blood.

6. The method for reprogramming human cells into induced pluripotent stem cells according to any one of claims 1 to 5, characterized in that: The following steps are involved: The three transcription factors OCT4, SOX2 and LIN28 are transferred into somatic cells, induced using an induction medium containing the chemical inducer described in claim 1, and finally cultured using a pluripotent stem cell medium until the induced pluripotent stem cell clones are fully mature, and then randomly picking clones for amplification; Wherein, the induction medium is supplemented with 2-4 μM chir99021, 80-120 ng / ml FGF2, 200-300 μM NaB, 1-3 μM SB203580, 0.5-1.5 μM peficitinib, 0.05-0.2 μM VTP50469, 1-5 μM FPFT-2216, 1-3% B27 by volume, 1% N2 by volume pluripotent stem cell culture medium; The pluripotent stem cell culture medium is mTeSR1 culture medium.

7. The method for reprogramming human cells into induced pluripotent stem cells according to claim 6, characterized in that: The three transcription factors OCT4, SOX2 and LIN28 are transferred into somatic cells by electrofection of episomal plasmids. The episomal plasmid is a pCPE4 vector carrying the three transcription factors after modification, wherein the two transcription factors OCT4 and SOX2 are constructed on the same pCPE4 vector.

8. The method for reprogramming human cells into induced pluripotent stem cells according to claim 7, characterized in that: After electroporation, peripheral blood expansion medium was immediately added and the cells were seeded in well plates coated with basement membrane matrix and cultured for 46 to 50 hours; Add induction medium and continue culturing for 46 to 50 hours. Then, add new induction medium by incomplete medium replacement and continue induction for 46 to 50 hours. Repeat the operation 1 to 3 times, discard all the old medium, add pluripotent stem cell medium and culture. Replace the pluripotent stem cell medium every day until the induced pluripotent stem cell clones are fully mature. Pick clones for expansion at any time.

9. The method for reprogramming human cells into induced pluripotent stem cells according to claim 8, characterized in that: The peripheral blood expansion medium is a serum-free medium supplemented with SCF 80-120 ng / mL, FLT-3 80-120 ng / mL, IL-3 10-30 ng / mL, IL-6 10-30 ng / mL, and TPO 80-120 ng / mL.

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