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JP-7854685-B2 - Method for producing naive human iPS cells from somatic cells

JP7854685B2JP 7854685 B2JP7854685 B2JP 7854685B2JP-7854685-B2

Inventors

  • 國富 晃
  • 朱 亜峰
  • 川口 実太郎

Assignees

  • 国立大学法人京都大学
  • 株式会社ＩＤファーマ

Dates

Publication Date
20260507
Application Date
20211224
Priority Date
20201225

Claims (20)

  1. A method for producing naive induced pluripotent stem cells from human somatic cells, (1) A step of introducing one or more vectors containing reprogramming factors into human somatic cells. (2) A step of culturing the somatic cells in the presence of a naive medium to obtain cultured cells , and (3) A step in which the cultured cells are cultured under different conditions than those in step (2), wherein the different conditions are such that the amount of vector per cultured cell is reduced to 30% or less compared to the start of step (3). A method comprising, wherein one or more vectors are selected from Sendai virus vectors containing the TS12 mutation (TS mutation (G69E/T116A/A183S mutation in M protein, A262T/G264R/K461G mutation in HN protein, L511F mutation in P protein, and N1197S/K1795E mutation in L protein) in addition to the D433A/R434A/K437A mutation in P protein), the TS7 mutation (TS mutation in addition to the Y942H/L1361C/L1558I mutation in L protein), or the TS15 mutation (TS mutation in addition to the D433A/R434A/K437A mutation in P protein and L1361C/L1558I mutation in L protein) .
  2. The method according to claim 1 , wherein step (2) is started 1 to 10 days after step (1).
  3. The method according to claim 1 or 2 , wherein step (2) is performed for 1 to 20 days.
  4. The method according to any one of claims 1 to 3 , wherein step (3) includes culturing the cultured cells at 38°C or higher.
  5. The method according to any one of claims 1 to 4 , wherein one or more vectors include vectors containing a target sequence of microRNA specific to induced pluripotent stem cells.
  6. The method according to claim 5 , wherein the target sequence of the microRNA is located in the coding region, 5'UTR, or 3'UTR of an NP gene or a P gene.
  7. The method according to claim 6 , wherein the microRNA is miR-367.
  8. The method according to any one of claims 1 to 7 , wherein the reprogramming factor comprises the OCT gene, the SOX gene, the MYC gene, and/or the KLF gene.
  9. The method according to claim 8 , wherein the one or more vectors include a vector containing the OCT gene, the SOX gene and the KLF gene, a vector containing the MYC gene and a vector containing the KLF gene.
  10. The method according to claim 9 , wherein the vector containing the MYC gene is a Sendai virus vector containing the TS15 mutation.
  11. The method according to claim 9 or 10 , wherein the vector comprising the OCT gene, the SOX gene, and the KLF gene, and the vector comprising the KLF gene, are Sendai virus vectors comprising the TS12 mutation.
  12. The method according to any one of claims 1 to 11 , wherein in step (3), the amount of the vector per cultured cell decreases to 30% or less compared to the amount at the start of step (3) within 12 days from the start of step (3) .
  13. The method according to any one of claims 1 to 12 , wherein the naive culture medium contains one or more compounds selected from LIF, MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors.
  14. The method according to claim 13 , wherein the naive culture medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo.
  15. The method according to any one of claims 1 to 14 , wherein steps (2) and (3) are carried out in the absence of feeder cells.
  16. The method according to any one of claims 1 to 15 , wherein the human somatic cell is a mononuclear cell or a fibroblast.
  17. A kit for producing naive induced pluripotent stem cells from human somatic cells, including the following: One or more vectors containing reprogramming factors, wherein the one or more vectors are selected from Sendai virus vectors containing TS12 mutations (TS mutations (G69E/T116A/A183S mutations in M protein, A262T/G264R/K461G mutations in HN protein, L511F mutations in P protein, and N1197S/K1795E mutations in L protein) in addition to the D433A/R434A/K437A mutation in P protein), TS7 mutations (TS mutations in addition to the Y942H/L1361C/L1558I mutation in L protein), or TS15 mutations (TS mutations in addition to the D433A/R434A/K437A mutation in P protein and L1361C/L1558I mutation in L protein) ; and naive medium.
  18. The kit according to claim 17 , wherein one or more vectors include vectors containing a target sequence of microRNA specific to induced pluripotent stem cells.
  19. The kit according to claim 18 , wherein the target sequence of the microRNA is located in the coding region, 5'UTR, or 3'UTR of an NP gene or a P gene.
  20. The kit according to claim 19 , wherein the microRNA is miR-367.

Description

This application relates to a method for producing naive induced pluripotent stem cells from human somatic cells. Mouse induced pluripotent stem (iPS) cells and embryonic stem (ES) cells are known to be naive, a highly undifferentiated state similar to that of a preimplantation blastocyst, while human iPS cells and human ES cells produced by conventional methods are known to be primed, a more advanced developmental state similar to that of a postimplantation embryo. Compared to naive mouse iPS cells, human iPS cells have been criticized for having limited pluripotency, being difficult to genetically manipulate, and exhibiting greater variability in gene expression and pluripotency among cell lines. One of the main reasons for these issues is that human iPS cells are more advanced in development than naive cells, being in a primed state. Therefore, a method for producing human pluripotent stem cells with naive characteristics is urgently needed for the advancement of regenerative medicine. To date, the following methods for producing human naive pluripotent stem cells have been reported: (1) a method of converting primed pluripotent stem cells into naive pluripotent stem cells by expressing a specific gene and culturing them in a culture medium containing a specific component (Patent Document 1, Non-Patent Document 1); (2) a method of converting primed pluripotent stem cells into naive pluripotent stem cells by culturing them in a culture medium containing a specific component (Patent Documents 2-4); and (3) a method of producing naive iPS cells by culturing somatic cells (fibroblasts) in a culture medium containing a specific component from a certain point during the reprogramming process (Non-Patent Documents 2-4). WO2016/148253Japanese Patent Publication No. 2015-136349WO2016/179243WO2017/170849 Takashima Y et al., Cell, 158:1254-1269, 2014Theunissen T et al., Cell Stem Cell, 15:471-487, 2014Kilens S et al., Nature Communications, 9:360-, 2018Liu X et al., Nature Methods, 14:1055-, 2017 Structure of the Sendai virus vector (CytoTune-iPS2.0L) conventionally used to produce naive iPS cells from somatic cells.A protocol for producing naive iPS cells from human dermal fibroblasts (HDFs). The vertical bars indicate an example of the timing for changing the culture medium.Phase-contrast microscopy image of HDF-derived naive iPS cells that have been passaged 45 times.Immunostaining images of pluripotency markers in HDF-derived naive iPS cells that have been passaged 45 times. The three images side-by-side are from the same field of view.The effect of high-temperature culture on the genome quantity of Sendai virus vectors.A protocol for producing naive iPS cells from human peripheral blood mononuclear cells (PBMCs). The vertical bars indicate an example of the timing of medium changes.Quantitative evaluation of the genomic amount of Sendai virus vector in PBMC-derived naive iPS cells, per passage from day 14.Principal component analysis integrating single-cell data from each cell type.Expression patterns of imprinted genes and X chromosome-related genes in each cell type.Density bean plot showing global methylation across different cell types.Principal component analysis showing global methylation across different cell types.The percentage of 5-methylcytosine in total cytosine in HDF-derived naive iPS cells obtained in this example.Early differentiation potential of the three germ layer components of HDF or PBMC-derived naive iPS cells after long-term passage (P16-45). Reset Naive ESCs (in-house) were used as a control.Phase-contrast images and immunofluorescence staining for SeV in naive human iPSCs initialized with CytoTune-iPS 2.0L (nCT2.0L_1) 94 days after SeV infection. Scale bar represents 100 μm.qRT-PCR analysis of SeV genome expression in naive iPSCs initialized using CytoTune 2.0 or a 2.0L SeV initialization kit. Values are normalized by GAPDH expression levels and shown as mean ± s.d. Each point has n=3.qRT-PCR analysis of GAPDH-normalized SeV vector genome expression in naive human iPSCs initialized with CytoTune 2.0 or 2.0L. Data are shown as mean ± s.d. Each point has n=3. Primers and SYBR Green designed to specifically detect each SeV vector were used in this experiment.Schematic diagram of the SeV vector and modified initialization cocktail. The SeV18+KLF4/TSΔF(KLF4/TS) vector in CytoTune-2.0 or the 2.0L initialization kit was replaced with one of the following three modified vectors: SeV18+KLF4/TS12ΔF(KLF4/TS12), SeV18+KLF4/PmiR367T2/TSΔF(KLF4/miR/TS), or SeV18+KLF4/PmiR367T2/TS12ΔF(KLF4/miR/TS12). KLF4/miR/TS12 was newly developed in this embodiment.qRT-PCR analysis of hsa-miRNA367-3p expression in HDF and PSC normalized by hsa-miRNA423-3p expression. Data are shown as mean ± s.d. n=3. ND indicates no detection after 40 amplification cycles.Experimental design for inducing naive human iPSCs from HDF or PBMC using a modified SeV-OSKL cocktail, and for changes in incubation temperature after the ge