JP7813038B2 - Efficient method for generating induced pluripotent stem cells - Google Patents

Description

The present invention relates to a method for improving the efficiency of induced pluripotent stem (iPS) cell establishment by inhibiting the function of transcription enhancer-associated domain family member-3 (hereinafter also referred to as "TEAD3") in the nuclear reprogramming process of somatic cells, as well as an agent for improving the efficiency of iPS cell establishment comprising a TEAD3 function inhibitor. The present invention also relates to a method for producing iPS cells by introducing a nuclear reprogramming substance and a TEAD3 function inhibitor into somatic cells.

Somatic cell reprogramming, driven by nuclear reprogramming factors such as Yamanaka factors (Oct3/4, Sox2, Klf4 (and c-Myc)), must overcome a set of powerful transcription factors that protect somatic cell identity in order to reprogram cells and dedifferentiate them into iPS cells. To improve the efficiency of iPS cell establishment, numerous studies have focused on identifying key factors that act as reprogramming barriers. Nevertheless, reprogramming remains an inefficient process.

The somatic cell reprogramming process can be broadly divided into initiation, stabilization, and maturation stages, and is coupled with the execution of multiple biological programs, including major changes in transcriptional networks. p53 is widely known to act as a critical barrier to reprogramming. Indeed, downregulation of p53 has been shown to significantly increase the number of iPS colonies in mouse and human fibroblasts (see, for example, Patent Document 1 and Non-Patent Document 1). However, because p53 acts as a defense mechanism against uncontrolled cell proliferation in response to DNA damage, some have pointed out that downregulation of the p53 pathway may result in genomic instability and pose safety concerns. Transient p53 suppression using a non-integrating plasmid offers a safer method for improving iPS cell establishment efficiency (see, for example, Non-Patent Document 2).

The present inventors previously reported that inhibiting p38 function during the nuclear reprogramming process of somatic cells can improve the efficiency of iPS cell establishment (Patent Document 2). However, the underlying mechanism remains poorly understood.

International Publication No. 2009/157593 International Publication No. 2012/036299

Hong, H. et al. (2009) Nature 460, 1132-1135. Okita, K. et al. (2011) Nat Methods 8,409-412.

The objective of this invention is to identify a novel key factor that acts as a reprogramming barrier for somatic cells, and to improve the efficiency of iPS cell establishment by controlling this factor to disable the reprogramming barrier.

To achieve the above objectives, the present inventors systematically investigated the role of p38 MAPK inhibitors and found that p38 inhibition can dramatically improve the efficiency of human iPS cell establishment at both the initiation and stabilization stages of reprogramming.
The present inventors also found that dual inhibition of both the p38 and p53 pathways during reprogramming further increased the efficiency of iPS colony formation compared with inhibition of either pathway alone. Among the genes whose expression was significantly downregulated by dual inhibition, the inventors identified TEAD3 as a gene whose expression could improve iPS cell establishment efficiency to a similar extent as that achieved by dual inhibition, leading to the completion of the present invention.

That is, the present invention is as follows.
[1] A method for improving the efficiency of establishing induced pluripotent stem (iPS) cells, the method comprising inhibiting the function of transcription enhancer-associated domain family member-3 (TEAD3) in the nuclear reprogramming process of somatic cells.
[2] The following (a) to (c):
(a) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the TEAD3 gene;
The method according to [1], wherein the function of TEAD3 is inhibited by introducing into somatic cells either (b) an antisense nucleic acid against a transcription product of the TEAD3 gene; or (c) a ribozyme nucleic acid against a transcription product of the TEAD3 gene.
[3] The method according to [1], wherein the function of TEAD3 is inhibited by introducing a dominant-negative mutant of TEAD3 or a nucleic acid encoding the mutant into somatic cells.
[4] The method according to [1], wherein the function of TEAD3 is inhibited by inhibiting a transcription coactivator of TEAD3 in somatic cells.
[5] (a) or (b) below:
(a) a decoy nucleic acid against TEAD3;
(b) The method according to [1], wherein the function of TEAD3 is inhibited by introducing into somatic cells an oligonucleic acid containing a nucleotide sequence represented by rrrcwwgyyynnnnnnnnnnnnnrrrcwwgyyy (r represents a or g, w represents a or t, y represents c or t, and n each independently represents none, a, g, t, or c; SEQ ID NO: 3).
[6] An agent for improving iPS cell establishment efficiency, comprising a substance that inhibits the function of TEAD3.
[7] The inhibitor is one of the following (a) to (c):
(a) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the TEAD3 gene;
The agent according to [6], which is either (b) an antisense nucleic acid against a transcription product of the TEAD3 gene; or (c) a ribozyme nucleic acid against a transcription product of the TEAD3 gene.
[8] The agent described in [6], wherein the inhibitor is a dominant-negative mutant of TEAD3 or a nucleic acid encoding the same.
[9] The agent described in [6], wherein the inhibitor is an inhibitor of a TEAD3 transcription coactivator.
[10] The inhibitor is (a) or (b) below:
(a) a decoy nucleic acid against TEAD3;
(b) The agent according to [6], which is an oligonucleic acid comprising a nucleotide sequence represented by rrrcwwgyyynnnnnnnnnnnnnrrrcwwgyyy (r represents a or g, w represents a or t, y represents c or t, and n each independently represents none, a, g, t, or c; SEQ ID NO: 3).
[11] A method for producing iPS cells, comprising contacting somatic cells with a nuclear reprogramming substance and a substance that inhibits the function of TEAD3.
[12] The inhibitor is one of the following (a) to (c):
(a) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the TEAD3 gene;
The method according to [11], wherein the nucleic acid is either (b) an antisense nucleic acid against a transcription product of the TEAD3 gene; or (c) a ribozyme nucleic acid against a transcription product of the TEAD3 gene.
[13] The method according to [11], wherein the inhibitor is a dominant-negative mutant of TEAD3 or a nucleic acid encoding the same.
[14] The method according to [11], wherein the inhibitor is an inhibitor of a TEAD3 transcription coactivator.
[15] The inhibitor is (a) or (b) below:
(a) a decoy nucleic acid against TEAD3;
(b) The method according to [11], wherein the oligonucleic acid comprises a nucleotide sequence represented by rrrcwwgyyynnnnnnnnnnnnnrrrcwwgyyy (r represents a or g, w represents a or t, y represents c or t, and n each independently represents none, a, g, t, or c; SEQ ID NO: 3).
[16] The method according to any one of [11] to [15], wherein the nuclear reprogramming substances are Oct3/4, Klf4 and Sox2, or nucleic acids encoding the same.
[17] The method according to any one of [11] to [15], wherein the nuclear reprogramming substances are Oct3/4, Klf4, Sox2, and c-Myc, L-Myc, or N-Myc, or nucleic acids encoding the same.

The present invention significantly improves the efficiency of establishing iPS cells from somatic cells.

Figure 1 shows the effect of small-molecule p38 inhibitors on mouse iPS cell establishment. A. To examine the effect of a p38 inhibitor (SB202190; 10 μM) on seven compounds (VC, 10 μg/mL; VA, 1.9 mM; CH, 3 μM; PD, 0.5 μM; IL, 0.2 ng/mL; AS, 5 μM; RA, 1 μM) at concentrations proven to alter MEF reprogramming efficiency in vitro, iPS cell colony formation assays were performed. Primary MEFs ectopically expressing four factors (4F; Oct3/4, Sox2, Klf4, and c-Myc) inserted into vectors with a Nanog-GFP cassette as the final effector, a reporter of pluripotency, were individually assayed for induction of reprogramming. To evaluate the reprogramming efficiency of each compound alone, the number of GFP-positive (GFP ⁺ ) colonies was counted 21 days (Day 21) (left bar) and 28 days (Day 28) (right bar) after treatment and compared with the number of GFP-positive colonies in the 4F-transduced negative control (DM). B. Schematic representation of four SB202190 treatment protocols (A-D). The time axis indicates the period of each treatment, with Day 0 representing the day the four factors were transduced into MEFs (A: Days 1-4, B: Days 1-8, C: Days 8-16, D: Days 1-16). C. The reprogramming efficiency of each treatment was examined on Days 21 and 28, and the number of iPS cell colonies induced by 4F and the p38 inhibitor (SB202190) (lower bar) was compared with the number of iPS cell colonies induced by 4F alone treated with vehicle (DMSO) (upper bar). D. F. Fall plot showing the difference in the number of GFP ⁺ colonies between Day 21 and Day 28 for the experimental group treated with SB202190 during Period A. E. Fall plot showing the difference in the number of GFP ⁺ colonies on Day 21 and Day 28 between the experimental group treated with SB202190 during Period A and the experimental group treated with SB202190 during Period D. Figure 1 shows the effect of small-molecule p38 inhibitors on human iPS cell establishment. A. Schematic representation of four p38 inhibitor treatment protocols (Periods A to D). The time axis indicates the period of each treatment, with Day 0 representing the day the four factors were introduced into HDFs (A: Days 2-4, B: Days 6-20, C: Days 20-32, D: Days 6-32). B. The effect of p38 inhibitors on HDF reprogramming efficiency was examined using an iPS cell colony formation assay. Primary HDFs ectopically expressing four factors (4F; Oct3/4, Sox2, Klf4, and c-Myc) were treated with 10 μM SB202190, SB203580, or SB239063 using Protocols A or D. On days 16, 24, and 32, the reprogramming efficiency was measured by counting the number of ES cell-like (iPS cell) colonies and comparing them with HDFs reprogrammed with 4F alone (DMSO) without p38 inhibitor treatment. The left graph shows the results of inhibitor treatment during period A, and the right graph shows the results of inhibitor treatment during period D. For each day, the results are shown for SB239063, SB203580, SB202190, and DMSO treatment, respectively. ^* p<0.05, ^** p<0.01. C. Phase-contrast images of human ES cell-like colonies induced from HDFs with 4F + SB202190. D. Alkaline phosphatase staining assay for iPS cells induced from HDFs with 4F alone (DMSO) or 4F + SB202190 (SB202190). E. Representative immunohistochemical staining images for pluripotency markers (OCT4, SOX2, and TRA-1-60) in iPS cell colonies induced from HDFs by 4F + SB202190 (right panel). The left panel shows nuclear staining with DAPI. Scale bar: 100 μm. F. The effect of the p38 inhibitor (SB202190) on the reprogramming efficiency of HDFs induced by the introduction of three factors (3F; Oct3/4, Sox2, and Klf4) was examined using an iPS cell colony formation assay. Reprogramming efficiency was measured as the number of ES cell-like (iPS cell) colonies on Day 24 (bottom) and Day 32 (top). The number of iPS cell colonies induced from HDFs by 3F + SB202190 (upper bar for each day) was compared with the number of iPS cell colonies induced by 3F and vehicle (DMSO) (lower bar for each day). ^* p<0.05. G. The effect of the p38 inhibitor (SB202190) on the reprogramming efficiency of HDFs induced by the introduction of reprogramming factors via an episomal vector (pCXLE) was examined using an iPS cell colony formation assay. Reprogramming efficiency was measured as the number of ES cell-like (iPS cell) colonies on Day 24 (bottom) and Day 32 (top). The number of iPS cell colonies induced from HDFs by reprogramming factors plus SB202190 (upper bar for each day) was compared with the number of iPS cell colonies induced by reprogramming factors plus vehicle (DMSO) (lower bar for each day). ^* p<0.05, ^*** p<0.001 Figure 1 shows pluripotency and genomic stability in human iPS cells established using a small-molecule p38 inhibitor. A. qRT-PCR analysis of the expression of pluripotency genes (OCT4, SOX2, and NANOG), indicators of somatic cell reprogramming. Values are relative to the expression level in ES cells, with expression levels set at 1. SB1-SB3: human iPS cell clones induced by SB202190 treatment; DM and B7: human iPS cell clones induced with 4F alone; ES: human ES cells; HD: HDF. B. Karyotype analysis of human iPSC clones induced by SB202190 treatment. C. In vitro differentiation assay of human iPSC clones induced by SB202190 treatment. From left to right, differentiation into embryoid bodies (EBs), ectodermal lineages (β-III-tubulin positive), mesodermal lineages (α-SMA positive), and endodermal lineages (AFP positive) are shown. Nuclei were stained with Hoechst 33342. Scale bar: 100 μm. D. Teratoma formation assay of human iPSC clones induced by SB202190 treatment. The left panel shows a schematic diagram of the three germ layer lineages (ectoderm, mesoderm, and endoderm). The stained photographs show differentiation into the ectoderm, mesoderm, and endoderm lineages, respectively, from left to right. E. Left panel: The number of iPS cell colonies induced by 4F transfection and p38 inhibitor (SB202190) treatment (upper bar for each HDF) from four different donors (HDF1616, HDF1079, HDF1078, and Tig109) was compared with the number of iPS cell colonies induced by 4F transfection and vehicle (DMSO) treatment (lower bar for each HDF) using an iPS cell colony formation assay. Right panel: Statistical analysis of iPS cell colony formation efficiency based on results from four donors (by t-test; ^* p<0.05). Figure 1 shows the results of transcriptome analysis of human iPS cells established by p38 and/or p53 inhibition. A. Comparison of p53 mRNA levels in HDFs reprogrammed with 4F alone (DMSO) and clones reprogrammed with 4F and p53 shRNA (shp53). The expression level of the former is set to 1, and the relative values are shown. ^*** p<0.001. B. The number of iPS cell colonies induced from HDFs by 4F introduction and p53 inhibition (shp53) and/or p38 inhibition (SB202190) was compared with the number of iPS cell colonies induced by 4F introduction and vehicle treatment (DMSO) using an iPS cell colony formation assay. ^** p<0.01, ^*** p<0.001. C. Principal component analysis (PCA) of over 58,000 genes expressed in the four groups correlated with increased iPSC colony number (△). D. Top panel: Agglomerative hierarchical clustering of samples. Distances between samples were calculated by applying Manhattan distance and creating a dendrogram using the agnes function. Bottom panel: Sample correlation matrix showing transcriptome differences obtained by consensus clustering of mRNA data. E. Scatter plot of clustering results using the k-means algorithm. F. Heat map of differentially expressed genes (DEG) analysis. 1147 DEGs between clusters A and C (left) and 2185 DEGs between clusters A and B (right) are shown. This figure shows the results of a search for genes whose expression levels are specifically reduced when both the p38 pathway and the p53 pathway are inhibited. A. Genes (DEGs) whose expression levels were reduced in iPS cells compared to HDFs were extracted and compared when p53 was inhibited alone (shp53) and when p53 and p38 were dually inhibited (shp53+SB202190) in conjunction with 4F introduction. 651 DEGs whose expression levels were reduced only with dual inhibition were found (top). Of these, 149 DEGs showed a two-fold or greater decrease in expression (bottom). B. Genes (DEGs) whose expression levels were reduced in iPS cells compared to HDFs were extracted and compared when p38 was inhibited alone (SB202190) and when p53 and p38 were dually inhibited (shp53+SB202190) in conjunction with 4F introduction. 1,056 DEGs whose expression levels were reduced only with dual inhibition were found (top). Of these, 145 DEGs showed a two-fold or greater decrease in expression (bottom). C. The overlap between the 651 genes whose expression levels were reduced only by p53/p38 dual inhibition compared to p53 alone and the 1,056 genes whose expression levels were reduced only by p53/p38 dual inhibition compared to p38 alone was examined. 340 genes were found to overlap. D. Of the 340 genes extracted in C, 31 were classified as having transcription factor or DNA binding activity. E. Analysis of significant GO terms related to biological processes for DEGs whose expression levels were commonly reduced by p53/p38 dual inhibition. Figure 1 shows the effect of TEAD3 inhibition on human iPS cell establishment and transcriptional regulation of the TEAD3 gene by Klf4. A. The relative expression levels of TEAD3 are shown when 4F is introduced into HDFs (DMSO), or when 4F is introduced and p38 is inhibited alone (p382KO), p53 is inhibited alone (shp53), or p53 and p38 are inhibited together (shp53+p382KO). +100 on the X-axis indicates the expression level of DMSO, and -100 on the X-axis indicates no expression. For example, -75 on the X-axis indicates an expression level 0.125 times that of DMSO. Dashed arrows indicate an increase in the number of iPS cell colonies in that direction. B. Figure 1 shows TEAD3 protein expression in two types of HDF (HDF1616, HDF1079) after transfection with 4F alone (4F), 4F with a nonspecific shRNA (Scr), or 4F with two TEAD3 shRNAs (4F-shTEAD3#1 and 4F-shTEAD3#2). In both HDFs, TEAD3 protein expression was significantly suppressed by the two TEAD3 shRNAs. C. Figure 1 shows TEAD3 mRNA expression in two types of HDF (HDF1616, HDF1079) after transfection with 4F alone (4F), 4F with a nonspecific shRNA (Scr), or 4F with two TEAD3 shRNAs (4F-shTEAD3#1 and 4F-shTEAD3#2). In both HDFs, TEAD3 mRNA expression was significantly suppressed by the two TEAD3 shRNAs. ^** p<0.01, ^*** p<0.001. D. The numbers of iPS cell colonies obtained when 4F alone (4F), 4F with a nonspecific shRNA (Scr), or 4F with two TEAD3 shRNAs (4F-shTEAD3#1 and 4F-shTEAD3#2) were transfected into two types of HDF (HDF1616 and HDF1079). ^* p<0.05, ^** p<0.01E. Alkaline phosphatase staining images (left: whole dish; right: enlarged photograph) of iPS cell colonies obtained when 4F alone (4F), 4F with a nonspecific shRNA (Scr), or 4F with two TEAD3 shRNAs (4F-sh#1 and 4F-sh#2) were transfected into HDF. F. Changes in TEAD3 expression during the MEF reprogramming process at early (day 2 and day 4), intermediate (day 8), and late (day 18) phases, as well as in iPSCs, are shown. G. Figure 1 shows changes in TEAD3 expression in SSEA1-positive and SSEA1-negative cells during the MEF reprogramming process. H. The transcription start site (TSS) sequence of the TEAD3 gene predicted to bind KLF4 and its relative binding score are shown. I. Prediction of KLF4 binding consensus sequences based on analysis of cis-element binding sequences of target genes known to be bound by KLF4. A. Cell counts of mouse iPS cells (20D17; initial cell density: 1 x ¹⁰⁵ cells/well) 96 hours after SB202190 treatment ( ^** : p<0.01; compared with DMSO). B. Mouse iPS cells obtained by SB202190 treatment gave rise to chimeric mice with brown coat color. C. Germline transmission occurred in mice derived from mouse iPS cells obtained by SB202190 treatment. These results demonstrate that p38 inhibition promotes proliferation of HDFs and HDFs in the early phase of reprogramming, but does not affect the proliferation of human iPS cells. A. HDFs were seeded into 6-well plates at a density of 1 x ¹⁰⁵ cells/well and cultured for 96 hours with three p38 inhibitors. Total cell numbers were counted on days 2, 4, 6, and 8 after treatment. Results from three independent experiments are shown as means and standard deviations ( ^* : p<0.05; compared to DMSO). B. 4F-transfected HDFs were cultured for 96 hours with three p38 inhibitors. Total cell numbers were counted on days 2, 4, 6, and 8 after treatment. Results from three independent experiments are shown as means and standard deviations ( ^* : p<0.05; compared to DMSO). C. Human iPS cells (201B7) were seeded onto SNL feeder cells at a density of 2 x ¹⁰ cells/well and cultured for 96 hours with one of three p38 inhibitors. Total cell numbers were counted four days after treatment. Results from three independent experiments are shown as the mean and standard deviation. Correlation between TEAD3 expression and p53 expression in publicly available datasets (GSE36664 containing transcriptome data for MEFs, GSE45276 containing transcriptome data for human lung fibroblasts (HLFs), and a dataset containing transcriptome data for HDFs) is shown (R: Pearson correlation coefficient). Correlations between TEAD3 expression and p38δ, ERK4, p44-ERK1, CDK4, and CDK6 expression in publicly available single-cell RNA-Seq datasets from HDFs are shown (R: Pearson correlation coefficient). Figure 1. Evaluation of the causal role of TEAD3 in tumor initiation and cell cycle kinetics in HeLa cells. First, daughter cell lines (plenti/Ubc-Slc7a1) expressing the ecotropic receptor Slc7a1 were generated to enable retroviral infection, and shTEAD3 was retrovirally introduced. Clones with downregulated TEAD3 expression exhibited enhanced cell proliferation and produced larger tumor-like nodules, suggesting that TEAD3 plays a causal role in cervical cancer progression by enhancing cancer-inducing cellular properties ( ^* : p<0.05, ^** : p<0.01; compared with Scr (HeLa cells transfected with a nonspecific shRNA)). TEAD3 expression analysis in the human cervical cancer dataset GSE6791 (Cancer Res 2007 May 15;67(10):4605-19). Left: Box plots show corrected TEAD3 mRNA levels in normal (N) and cancer (T) tissue samples. Right: Patients in the GSE691 data shown in the box plots were individually classified according to increased TEAD3 expression levels. A. Representative bright-field images of putative iPS cell colonies (squares) 12 days after transfection of HDF1616 with 4F (scr) or 4F + shTEAD3. B. Representative bright-field images of putative iPS cell colonies (squares) 12 days after transfection of HDF1079 with 4F + shTEAD3. C. Growth curves of HDF1616 cells in the reprogramming process transfected with 4F (Scr), 4F + shTEAD3, or 4F + shTEAD3 + shp53 ( ^* : p<0.05; compared with Scr (HDF1616 transfected with 4F + nonspecific shRNA)). D. Growth curves of iPS cells established by transfecting HDF1616 with 4F (Scr), 4F + shTEAD3, or 4F + shTEAD3 + shp53). E. Growth curves of HDF1079 cells in the reprogramming process transfected with 4F (Scr), 4F + shTEAD3, or 4F + shTEAD3 + shp53 are shown ( ^** : p<0.01; compared with Scr (HDF1079 transfected with 4F + nonspecific shRNA)). F. Growth curves of iPS cells established by transfecting HDF1079 with 4F (Scr), 4F + shTEAD3, or 4F + shTEAD3 + shp53.

The present invention provides a method for improving iPS cell establishment efficiency by inhibiting TEAD3 function during the nuclear reprogramming process of somatic cells (hereinafter also referred to as the "improved method of the present invention"). The means for inhibiting TEAD3 function are not particularly limited, but a preferred method involves introducing a TEAD3 function inhibitor into somatic cells. Accordingly, the present invention also provides an agent for improving iPS cell establishment efficiency (hereinafter also referred to as the "agent of the present invention") comprising a TEAD3 function inhibitor. Furthermore, the present invention provides a method for producing iPS cells by introducing a nuclear reprogramming substance and a TEAD3 function inhibitor into somatic cells (hereinafter also referred to as the "production method of the present invention"; the "improved method of the present invention" and the "production method of the present invention" may be collectively referred to as the "method of the present invention").

(A) Somatic Cell Source Somatic cells that can be used as starting materials for producing iPS cells in the present invention may be any cells other than germ cells derived from mammals (e.g., humans, mice, monkeys, cows, pigs, rats, dogs, etc.), and include, for example, keratinizing epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the surface of the tongue), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), metabolic and storage cells (e.g., hepatocytes), luminal epithelial cells that form the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner ducts (e.g., blood Examples of somatic cells include endothelial cells, ciliated cells with transport capacity (e.g., airway epithelial cells), extracellular matrix-secreting cells (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), blood and immune system cells (e.g., peripheral blood mononuclear cells, umbilical cord blood, T lymphocytes), sensory cells (e.g., rod cells), autonomic nervous system neurons (e.g., cholinergic neurons), supporting cells of sensory organs and peripheral neurons (e.g., satellite cells), central nervous system neurons and glial cells (e.g., astrocyte glial cells), pigment cells (e.g., retinal pigment epithelial cells), and their precursor cells (tissue precursor cells). There are no particular limitations on the degree of cell differentiation or the age of the animal from which the cells are collected. Both undifferentiated precursor cells (including somatic stem cells) and terminally differentiated mature cells can be used as the source of somatic cells in the present invention. Examples of undifferentiated precursor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

While there are no particular limitations on the mammalian individual from which somatic cells are obtained, when the resulting iPS cells are to be used in human regenerative medicine, it is particularly preferable to obtain somatic cells from the patient himself or from another person with the same or substantially the same HLA type, in order to avoid rejection. Here, "substantially the same" HLA type refers to a match in HLA type sufficient to allow the transplanted cells to survive when cells obtained by inducing differentiation of iPS cells derived from the somatic cells, using immunosuppressants or other methods, are transplanted into a patient. For example, this includes cases where the major HLA loci (e.g., the three HLA loci HLA-A, HLA-B, and HLA-DR) are identical (hereinafter the same). Furthermore, when iPS cells are not to be administered (transplanted) to humans, for example, when they are used as a cell source for screening to assess a patient's drug sensitivity or the presence or absence of side effects, it is similarly preferable to obtain somatic cells from the patient himself or from another person with the same genetic polymorphisms correlated with drug sensitivity or side effects.

Prior to subjecting somatic cells isolated from mammals to the nuclear reprogramming process, they can be pre-cultured in a known medium suitable for the culture of the cell type. Examples of such media include, but are not limited to, minimum essential medium (MEM) containing approximately 5-20% fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, 199 medium, and F12 medium. When using an introduction reagent such as cationic liposomes to contact the cells with nuclear reprogramming substances and p38 function inhibitors (and, if necessary, other substances that improve iPS cell establishment efficiency, as described below), it may be preferable to switch to a serum-free medium to prevent a decrease in introduction efficiency.

(B) Functional inhibitors of TEAD3 TEAD3, the target molecule in this invention, is a member of the transcription enhancer-associated domain (TEAD) family. This transcription factor activates the transcription of target genes by binding to the coactivators YAP or TAZ, whose nuclear translocation is controlled by the Hippo signaling pathway.

As used herein, "TEAD3" refers to a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2. As used herein, proteins and peptides are written in accordance with the convention of peptide notation, with the left end being the N-terminus (amino terminus) and the right end being the C-terminus (carboxyl terminus).
"An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2" means
(a) the amino acid sequence of an ortholog of human TEAD3 consisting of the amino acid sequence set forth in SEQ ID NO: 2 in another warm-blooded animal (e.g., guinea pig, rat, mouse, chicken, rabbit, dog, pig, sheep, cow, monkey, etc.); or (b) the amino acid sequence of a natural allelic variant or genetic polymorphism of human TEAD3 consisting of the amino acid sequence set forth in SEQ ID NO: 2 or the ortholog of (a) above.
Preferably, TEAD3 is human TEAD3 consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a natural allelic variant or genetic polymorphism thereof. Examples of such genetic polymorphisms include, but are not limited to, the SNP in which Thr (ACG) at position 254 is replaced with Met (ATG), which is registered in dbSNP as rs35080860.

As used herein, the term "TEAD3 function inhibitor" refers to any substance that can inhibit (1) the function of the TEAD3 protein or (2) the expression of the TEAD3 gene. In other words, the term "TEAD3 function inhibitor" as used herein includes not only substances that act directly on the TEAD3 protein to inhibit its function, or substances that act directly on the TEAD3 gene to inhibit its expression, but also substances that act on factors involved in transcriptional activation by TEAD3, thereby inhibiting the function of the TEAD3 protein or the expression of the TEAD3 gene.

In the present invention, a "substance that inhibits TEAD3 gene expression" refers to a substance that acts at any stage of the TEAD3 gene, such as the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. Therefore, substances that inhibit TEAD3 expression include, for example, substances that inhibit TEAD3 gene transcription (e.g., antigenes), substances that inhibit the processing of initial transcription products into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (e.g., antisense nucleic acids, miRNA) or that degrade mRNA (e.g., siRNA, gapmer-type antisense nucleic acids, ribozymes, miRNA), and substances that inhibit the post-translational modification of initial translation products. While substances that act at any stage can be used, substances that bind complementarily to mRNA and inhibit translation into protein or degrade mRNA are preferred.

A preferred example of a substance that specifically inhibits translation of TEAD3 gene mRNA into protein (or degrades mRNA) is a nucleic acid that contains a nucleotide sequence complementary to the nucleotide sequence of the mRNA, or a part thereof.
A nucleotide sequence complementary to the nucleotide sequence of the mRNA of the TEAD3 gene refers to a nucleotide sequence that is sufficiently complementary to the target sequence of the mRNA to inhibit its translation (or cleave the target sequence) under physiological conditions. Specifically, for example, it refers to a nucleotide sequence that has 90% or more, preferably 95% or more, more preferably 97% or more, and particularly preferably 98% or more homology with a nucleotide sequence that is completely complementary to the nucleotide sequence of the mRNA (i.e., the nucleotide sequence of the complementary strand of the mRNA) in the overlapping region. The "nucleotide sequence homology" in the present invention can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions: expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3.

More specifically, a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the TEAD3 gene is a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 1. Here, "stringent conditions" refers to, for example, the conditions described in *Current Protocols in Molecular Biology*, John Wiley & Sons, pp. 6.3.1-6.3.6, 1999, such as hybridization in 6xSSC (sodium chloride/sodium citrate) at 45°C, followed by one or more washes at 0.2xSSC/0.1% SDS at 50-65°C. However, those skilled in the art can appropriately select hybridization conditions that provide equivalent stringency.

Preferred examples of mRNA of the TEAD3 gene include the mRNA of human TEAD3 (RefSeq Accession No. NM_003214.4) comprising the nucleotide sequence represented by SEQ ID NO: 1, or its orthologs in other warm-blooded animals, as well as their natural allelic variants or genetic polymorphisms.

"A portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the TEAD3 gene" is not particularly limited in length or position, as long as it can specifically bind to the mRNA of the TEAD3 gene and inhibit protein translation from the mRNA (or degrade the mRNA), but from the standpoint of sequence specificity, it should contain a portion complementary to the target sequence that is at least 10 bases long, preferably 15 bases long, and more preferably 19 bases long.

Specifically, preferred examples of nucleic acids containing a portion of a nucleotide sequence complementary to the nucleotide sequence of mRNA of the TEAD3 gene include any of the following (a) to (c):
(a) a nucleic acid or a precursor thereof having RNAi activity against the mRNA of the TEAD3 gene
(b) antisense nucleic acid against the mRNA of the TEAD3 gene
(c) a ribozyme nucleic acid for the mRNA of the TEAD3 gene

(a) Nucleic acid or precursor thereof having RNAi activity against TEAD3 gene mRNA In this specification, double-stranded RNA consisting of an oligo-RNA complementary to the TEAD3 gene mRNA and its complementary strand, so-called siRNA, is defined as being included in nucleic acids containing a nucleotide sequence complementary to the nucleotide sequence of the TEAD3 gene mRNA or a part thereof.

siRNAs can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)). Examples of siRNA target sequences include, but are not limited to, AA+(N) ₁₉ , AA+(N) ₂₁ , or NA+(N) ₂₁ (where N is any base). The location of the target sequence is also not particularly limited. The selected group of candidate target sequences is examined for homology to 16-17 base contiguous sequences in mRNAs other than the target using homology search software such as BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), to confirm the specificity of the selected target sequence. For example, when the target sequence is AA+(N) ₁₉ , AA+(N) ₂₁ , or NA+(N) ₂₁ (N is any base), for a target sequence whose specificity has been confirmed, a double-stranded RNA consisting of a sense strand having a 3'-terminal overhang of TT or UU at 19-21 bases after AA (or NA), and an antisense strand having a sequence complementary to the 19-21 bases and a 3'-terminal overhang of TT or UU may be designed as an siRNA. Furthermore, short hairpin RNA (shRNA), which is a precursor of siRNA, can be designed by appropriately selecting any linker sequence (e.g., about 5-25 bases) capable of forming a loop structure and linking the sense strand and antisense strand via the linker sequence.

The sequences of siRNA and/or shRNA can be searched using search software provided free of charge on various websites. Examples of such websites include, but are not limited to, the siDESIGN Center provided by Horizon Discovery Ltd. (https://horizondiscovery.com/en/products/tools/siDESIGN-Center) and the siRNA Target Finder provided by GenScript (https://www.genscript.com/tools/sirna-target-finder).

In a preferred embodiment, the siRNA and shRNA of the present invention comprise a nucleotide sequence complementary to a sequence consisting of at least 15 consecutive nucleotides within the region represented by nucleotide numbers 219 to 247 (AGCAACCAGCACAATAGCGTCCAACAGCT: SEQ ID NO: 4) or 1207 to 1235 (AGCATGACCATCAGCGTCTCCACCAAGGT: SEQ ID NO: 5) in the nucleotide sequence represented by SEQ ID NO: 1.

As used herein, microRNAs (miRNAs) that target the mRNA of the TEAD3 gene are also defined as nucleic acids that contain a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the TEAD3 gene, or a portion thereof. miRNAs are involved in the post-transcriptional regulation of gene expression by either binding complementarily to the target mRNA and suppressing mRNA translation or by degrading the mRNA.

miRNAs are first transcribed from the genes that encode them as primary-microRNAs (pri-miRNAs), which are then processed by Drosha into precursor-microRNAs (pre-miRNAs) of approximately 70 bases in length with a characteristic hairpin structure. These are then transported from the nucleus to the cytoplasm, where they are further processed by Dicer to become mature miRNAs, which are then incorporated into RISC and act on target mRNAs. Therefore, pre-miRNAs or pri-miRNAs, preferably pre-miRNAs, can be used as miRNA precursors.

miRNAs can be searched for using target prediction software provided free of charge on various websites. Examples of such websites include, but are not limited to, TargetScan (http://www.targetscan.org/vert_72/) published by the Whitehead Institute in the United States and DIANA-micro-T-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index) published by the Alexander Fleming Center for Biomedical Sciences in Greece. Alternatively, miRNAs targeting TEAD3 mRNA can be searched for using TarBase (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8/index), a database of miRNAs experimentally proven to act on target mRNAs published by the University of Cesarea and the Institut Pasteur. For example, among the miRNAs that matched the TEAD3 mRNA hits in the database, those that scored highly in the target prediction software include hsa-miR-106b-5p, hsa-miR-20a-5p, etc. Sequence information for these miRNAs and/or pre-miRNAs can be obtained using, for example, miRBase (http://www.mirbase.org/search.shtml), published by the University of Manchester, UK.

The nucleotide molecules constituting siRNA and/or shRNA, or miRNA and/or pre-miRNA may be natural RNA or DNA, but may contain various chemical modifications to improve stability (chemical and/or enzymatic) and specific activity (affinity with RNA). For example, to prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide constituting the antisense nucleic acid may be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. Furthermore, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide may be substituted with _-OR ( _R = _CH3 (2'-O- _Me ), _CH2CH2OCH3 (2'-O-MOE), _CH2CH2NHC (NH) _NH2 , _CH2CONHCH3 , _{CH2CH2CN , etc.} ). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position.

The sugar conformations of RNA are predominantly C2'-endo (S-form) and C3'-endo (N-form). In single-stranded RNA, these two conformations exist in equilibrium, but upon duplex formation, the N-form is fixed. Therefore, to confer strong binding ability to target RNA, RNA derivatives in which the sugar conformation is fixed to the N-form by bridging the 2' oxygen and 4' carbon, such as BNA (LNA) (Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, J.S. et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides, Nucleotides, Nucleic Acids, 22, 1619-21, 2003), may also be used.

siRNA can be prepared by synthesizing the sense and antisense strands of the target sequence on mRNA using an automated DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at approximately 90 to 95°C for approximately 1 minute, and then annealing them at approximately 30 to 70°C for approximately 1 to 8 hours. Alternatively, siRNA can be prepared by synthesizing shRNA, which serves as a precursor to siRNA, and then cleaving it using a dicer. miRNA and pre-miRNA can be synthesized using an automated DNA/RNA synthesizer based on their sequence information.

As used herein, nucleic acids designed to produce siRNA or miRNA against TEAD3 gene mRNA in vivo are also defined as being encompassed by nucleic acids containing a nucleotide sequence complementary to the nucleotide sequence of TEAD3 gene mRNA, or a portion thereof. Examples of such nucleic acids include expression vectors constructed to express the shRNA or siRNA, miRNA, or pre-miRNA described above. As shown in the Examples below, TEAD3 expression increases both in the initiation stage of the reprogramming process (e.g., within 3 days after introduction of the nuclear reprogramming substance) and in the stabilization stage (e.g., 2-3 weeks after introduction of the nuclear reprogramming substance). Therefore, it is considered desirable to sustain TEAD3 functional inhibition throughout the nuclear reprogramming process. The use of an expression vector is preferable because it allows for the long-term, sustained delivery of nucleic acids that inhibit TEAD3 expression to somatic cells.

shRNAs can be prepared by designing an oligo-RNA containing a nucleotide sequence linking the sense and antisense strands of a target mRNA sequence with a spacer sequence of sufficient length (e.g., 5–25 bases) to form a suitable loop structure, and synthesizing the oligo-RNA using an automated DNA/RNA synthesizer. shRNA expression vectors are available in tandem and stem-loop (hairpin) types. The former link an expression cassette for the sense strand of siRNA and an expression cassette for the antisense strand in tandem; each strand is expressed in the cell and annealed to form double-stranded siRNA (dsRNA). The latter, on the other hand, incorporates an shRNA expression cassette into a vector; the shRNA is expressed in the cell and processed by a dicer to form dsRNA. While Pol II promoters (e.g., the CMV immediate-early promoter) can also be used, Pol III promoters are commonly used to ensure accurate transcription of short RNAs. Pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters. A sequence of four or more consecutive Ts is also used as a transcription termination signal. Expression cassettes for miRNAs and pre-miRNAs can also be prepared in the same manner as for shRNAs.

The siRNA or shRNA, or miRNA or pre-miRNA expression cassette constructed in this manner is then inserted into a plasmid or viral vector. Examples of such vectors include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses, as well as animal cell expression plasmids. TEAD3 is deeply involved in gene expression regulation via the Hippo signaling pathway, which is also involved in maintaining homeostasis. Therefore, it is preferable to quickly restore its function after the reprogramming barrier is released and somatic cells dedifferentiate into iPS cells. Therefore, non-integrative transient expression vectors, such as adenovirus vectors and plasmids, are more preferred as expression vectors. Among these, the use of episomal vectors capable of autonomous extrachromosomal replication is preferred, as they can sustain the expression of nucleic acids that inhibit TEAD3 expression throughout the nuclear reprogramming process and are rapidly eliminated from the cells after iPS cell establishment. Specific methods using episomal vectors are disclosed in Yu et al., Science, 324, 797-801 (2009).

Examples of episomal vectors include vectors that contain sequences necessary for autonomous replication derived from EBV, SV40, etc. as vector elements. Specific vector elements necessary for autonomous replication are replication origins and genes encoding proteins that bind to the replication origin and control replication. For example, for EBV, these include the replication origin oriP and EBNA-1 gene, and for SV40, these include the replication origin ori and SV40 large T antigen gene.

(b) Antisense nucleic acid against mRNA of TEAD3 gene In the present invention, an "antisense nucleic acid against mRNA of TEAD3 gene" is a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a part thereof, and has the function of inhibiting protein synthesis by binding to the target mRNA to form a specific and stable double strand.
Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, other polymers with non-nucleotide backbones (e.g., commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers), or other polymers containing special linkages, provided that the polymer contains nucleotides with configurations that allow base pairing or base attachment as found in DNA or RNA. They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or DNA:RNA hybrids. They can also be unmodified polynucleotides (or unmodified oligonucleotides), those containing known modifications, such as those labeled, capped, or methylated, those containing analogs of one or more natural nucleotides, or those containing intramolecular nucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages, or sulfuric acid groups. Nucleosides may contain linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side groups such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (e.g., monosaccharides, etc.), intercurrent compounds (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), alkylators, or modified linkages (e.g., α-anomeric nucleic acids, etc.). The terms "nucleoside,""nucleotide," and "nucleic acid" as used herein include those containing not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also have modified sugar moieties, for example, one or more hydroxyl groups may be substituted with halogens, aliphatic groups, or converted to functional groups such as ethers or amines.

As mentioned above, antisense nucleic acids may be DNA or RNA, or may be DNA/RNA chimeras. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H, causing selective degradation of the target RNA. Therefore, in the case of antisense DNA that directs degradation by RNase H, the target sequence may be not only a sequence in mRNA, but also a sequence in the intron region of the initial translation product of the TEAD3 gene. Intron sequences can be determined by comparing the genomic sequence with the cDNA nucleotide sequence of the TEAD3 gene using homology search programs such as BLAST and FASTA.

The target region of the antisense nucleic acid of the present invention is not particularly limited in length, as long as hybridization of the antisense nucleic acid results in inhibition of protein translation. It may be the entire or partial sequence of the mRNA encoding the protein, ranging from as short as about 10 bases to as long as the entire sequence of the mRNA or initial transcription product. Considering ease of synthesis, antigenicity, intracellular internalization, and other issues, oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but are not limited to these. Specifically, preferred target regions of the TEAD3 gene include, but are not limited to, the 5'-end hairpin loop, 5'-end 6-base pair repeat, 5'-end untranslated region, translation initiation codon, protein-coding region, ORF translation termination codon, 3'-end untranslated region, 3'-end palindrome region, and 3'-end hairpin loop.

In one embodiment, the target region of the antisense nucleic acid of the present invention, similar to the above-mentioned siRNA, can be a sequence consisting of at least 15 consecutive nucleotides within the region represented by nucleotide numbers 219 to 247 or 1207 to 1235 in the nucleotide sequence represented by SEQ ID NO: 1. When a gapmer-type antisense nucleic acid is used, TEAD3 mRNA can be degraded within the target region by the action of RNase H, thereby achieving an effect equivalent to that of siRNA.

TEAD4, a paralog of TEAD3, is known to have a splice variant lacking the N-terminal DNA-binding domain. This is thought to be due to the change in the position of the start codon caused by exon 3 skipping (Nat Commun 7:11840 | DOI: 10.1038/ncomms11840). The DNA-binding domain is highly conserved within the TEAD family, and it is predicted that TEAD3 also has a splice variant lacking the DNA-binding domain. Therefore, by using antisense nucleic acids targeting the splice-promoting sequence within exon 3 or an adjacent intron, transcription of the splice variant lacking exon 3 can be promoted, generating a TEAD3 isoform beginning with Met at amino acid 112 in the amino acid sequence represented by SEQ ID NO: 2. This isoform binds to the coactivator YAP/TAZ but is unable to bind to the promoters of target genes, functioning as a dominant-negative mutant of TEAD3.

Furthermore, the antisense nucleic acid of the present invention may not only hybridize with the mRNA or initial transcription product of the TEAD3 gene and inhibit translation into protein, but may also bind to these genes, which are double-stranded DNA, to form a triplex and inhibit transcription into RNA (antigene).

The nucleotide molecules that make up the antisense nucleic acid may also be modified in the same way as the siRNAs described above to improve stability, specific activity, etc.

The antisense oligonucleotides of the present invention can be prepared by determining the target sequence of mRNA or early transcription products based on the cDNA sequence or genomic DNA sequence of the TEAD3 gene, and then synthesizing a complementary sequence using a commercially available automated DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.). Antisense nucleic acids containing the various modifications described above can also be chemically synthesized using known methods.

Alternatively, antisense nucleic acids can be incorporated into expression vectors and introduced into somatic cells, as in the case of siRNAs, etc., described above.

(c) a ribozyme nucleic acid for the mRNA of the TEAD3 gene
Other examples of nucleic acids containing a nucleotide sequence complementary to the nucleotide sequence of the TEAD3 gene mRNA, or a portion thereof, include ribozyme nucleic acids capable of specifically cleaving the mRNA within the coding region. In a narrow sense, the term "ribozyme" refers to RNA with enzymatic activity for cleaving nucleic acids. However, as used herein, the term encompasses DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and are known to include hammerhead and hairpin types. Hammerhead types exert their enzymatic activity with approximately 40 bases. By arranging several bases (totaling approximately 10 bases) adjacent to the hammerhead structure at both ends to be complementary to the desired cleavage site in the mRNA, they can specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When the TEAD3 mRNA itself is double-stranded, the target sequence can be made single-stranded by using a hybrid ribozyme containing an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when a ribozyme is used in the form of an expression vector containing DNA encoding it, a hybrid ribozyme can be further ligated with a modified tRNA sequence to promote the translocation of the transcript into the cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].

Nucleic acids containing a nucleotide sequence complementary to the nucleotide sequence of the TEAD3 gene mRNA, or a portion thereof, can be provided in specialized forms such as liposomes or microspheres, or in a form with other components attached. Examples of such attached forms include polycationic bodies such as polylysine, which neutralize the charge of the phosphate backbone, and hydrophobic lipids (e.g., phospholipids, cholesterol, etc.) that enhance interaction with cell membranes and increase nucleic acid uptake. Preferred lipids for attachment include cholesterol and its derivatives (e.g., cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3' or 5' end of the nucleic acid via the base, sugar, or intramolecular nucleoside bond. Other groups include capping groups specifically placed at the 3' or 5' end of the nucleic acid to prevent degradation by nucleases such as exonucleases and RNases. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol, tetraethylene glycol, and the like.

When the nucleic acid containing a portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the TEAD3 gene is in the form of RNA, it can be introduced into somatic cells by techniques such as lipofection and microinjection.
On the other hand, when the vector is in the form of an expression vector containing DNA encoding the RNA, it can be introduced into cells by known methods depending on the type of vector. For example, in the case of a viral vector, a plasmid containing the DNA is introduced into appropriate packaging cells (e.g., Plat-E cells) or complementation cell lines (e.g., 293 cells), the viral vector produced in the culture supernatant is collected, and the vector is then used to infect cells using an appropriate method for each viral vector. For example, specific methods using retroviral vectors as vectors are disclosed in WO2007/69666, Cell, 126, 663-676 (2006), and Cell, 131, 861-872 (2007), and methods using lentiviral vectors as vectors are disclosed in Science, 318, 1917-1920 (2007). Methods using adenoviral vectors are described in Science, 322, 945-949 (2008).
On the other hand, in the case of a plasmid vector, which is a non-viral vector, the vector can be introduced into cells using the lipofection method, liposome method, electroporation method, calcium phosphate co-precipitation method, DEAE-dextran method, microinjection method, gene gun method, etc.

(d) an oligonucleotide containing a consensus binding sequence for p53;
Another preferred embodiment of the substance inhibiting TEAD3 gene expression is a substance that inhibits the binding of a transcriptional activator of the TEAD3 gene to the promoter region of the TEAD3 gene. For example, such a substance can include an oligonucleic acid containing the p53 consensus binding sequence rrrcwwgyyynnnnnnnnnnnnnnnrrrcwwgyyy (r represents a or g, w represents a or t, y represents c or t, and each n independently represents absent, a, g, t, or c; SEQ ID NO: 3). Preferably, the oligonucleic acid is double-stranded DNA. Since p53 is thought to positively regulate TEAD3 gene transcription by binding to cis-element sequences present in the TEAD3 promoter region, an oligonucleic acid containing the p53 consensus binding sequence can inhibit p53 binding to the TEAD3 promoter region and thereby suppress its transcription. The length of the oligonucleic acid is, for example, 20 to 50 nucleotides, preferably 25 to 40 nucleotides.

The oligonucleic acid can be produced by synthesizing the sense and antisense strands using a commercially available automated DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.) based on the sequence information of SEQ ID NO: 3 and annealing them.

The oligonucleic acid can be introduced into somatic cells by techniques such as lipofection or microinjection.

In the present invention, a "substance that suppresses the function of TEAD3" refers to any substance that suppresses the function of TEAD3 once it has been functionally produced (for example, the transcription activation function of a group of genes that maintain somatic cell identity). Examples include substances that bind to TEAD3 and suppress said function, substances that inhibit the binding activity of TEAD3 to target genes, and substances that inhibit the interaction between TEAD3 and transcriptional coactivators.

(e) Dominant-negative mutant of TEAD3
In humans, TEAD3 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The region from approximately 30 to 100 amino acids from the N-terminus is a DNA-binding domain highly conserved within the TEAD family, and the regions from approximately 200 onward are the YAP/TAZ-binding and transcriptional activation domains. Therefore, TEAD3 fragments lacking the DNA-binding domain can competitively bind to the transcriptional coactivators YAP/TAZ with endogenous full-length TEAD3 and suppress the transcriptional activation of target genes through the interaction between TEAD3 and YAP/TAZ. On the other hand, TEAD3 fragments lacking the YAP/TAZ-binding and transcriptional activation domains can competitively bind to the promoter regions of target genes with endogenous full-length TEAD3 and suppress the transcriptional activation of target genes through the interaction between TEAD3 and YAP/TAZ.

Dominant-negative mutants of TEAD3 can be obtained, for example, by designing an appropriate primer set and using (RT-)PCR to clone a nucleic acid encoding a TEAD3 fragment lacking the desired DNA-binding domain or YAP/TAZ-binding domain (transcription activation domain) from mRNA, cDNA, or a cDNA library derived from cells or tissues expressing TEAD3 from a warm-blooded animal such as a human, inserting the nucleic acid into an appropriate expression vector, introducing the vector into host cells, culturing the cells, and recovering the recombinant protein from the resulting culture.

Contact of dominant-negative mutants with somatic cells can be carried out using known methods for protein transfection into cells. Examples of such methods include methods using protein transfection reagents, methods using protein transduction domain (PTD) fusion proteins, and microinjection. Commercially available protein transfection reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systems), Pro-Ject ^™ Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), lipid-based Profect-1 (Targeting Systems), and membrane-permeable peptide-based Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif). Transfection can be performed according to the protocols provided with these reagents, but the general procedure is as follows: A dominant-negative mutant of p38 is diluted in an appropriate solvent (e.g., a buffer such as PBS or HEPES), added with the transfection reagent, and incubated at room temperature for 5–15 minutes to form a complex. This complex is then added to cells in serum-free medium and incubated at 37°C for one to several hours. The medium is then removed and replaced with serum-containing medium.

PTDs have been developed using the cell passage domains of proteins such as Drosophila-derived AntP, HIV-derived TAT, and HSV-derived VP22. A fusion protein expression vector incorporating the cDNA of a dominant-negative mutant of p38 and a PTD sequence is constructed and recombinantly expressed, and the fusion protein is recovered and used for transfection. Transfection can be performed in the same manner as above, except that no protein transfection reagent is added. This method is suitable for transfecting deletion mutants with relatively small molecular weights, such as p38DD.

Microinjection is a method in which a protein solution is placed into a glass needle with a tip diameter of approximately 1 μm and then punctured into a cell, ensuring reliable introduction of proteins into cells.

(f) Nucleic Acids Encoding Dominant-Negative Mutants of TEAD3 However, considering the ease of introduction into somatic cells, it is preferable to use dominant-negative mutants of TEAD3 in the form of nucleic acids encoding them rather than as the protein itself. Therefore, in another preferred embodiment of the present invention, the TEAD3 function inhibitor is a nucleic acid encoding a dominant-negative mutant of TEAD3. The nucleic acid may be DNA, RNA, or a DNA/RNA chimera, but is preferably DNA. Furthermore, the nucleic acid may be double-stranded or single-stranded. cDNA encoding a dominant-negative mutant of TEAD3 can be cloned by the techniques described above for producing the mutant protein.

The isolated cDNA, like the nucleic acid containing a portion of the nucleotide sequence complementary to the nucleotide sequence of the mRNA of the TEAD3 gene described above, can be inserted into an appropriate viral or non-viral expression vector and introduced into somatic cells using a similar gene transfer method.

(g) Inhibitors of TEAD3 Transcriptional Coactivators As mentioned above, TEAD3 activates the transcription of target genes by conjugating with the coactivators YAP/TAZ. When YAP/TAZ is phosphorylated (e.g., phosphorylated at Ser127), it binds to 14-3-3, localizes to the cytoplasm, and is inactivated. However, upon dephosphorylation, it dissociates from 14-3-3, translocates to the nucleus, and conjugates with TEAD3 to promote the transcription of target genes. Therefore, inhibiting the TEAD3 transcriptional coactivators YAP/TAZ can inhibit TEAD3 function.

Methods for inhibiting YAP/TAZ include introducing siRNA or miRNA against YAP or TAZ, or their precursors, antisense nucleic acids against YAP or TAZ, ribozyme nucleic acids, etc. into somatic cells to suppress their expression, or promoting phosphorylation of YAP/TAZ or complex formation with 14-3-3 to suppress its activation and nuclear translocation.

siRNA or shRNA against YAP or TAZ can be appropriately designed using the same method as described for siRNA against TEAD3, etc., based on the sequence information of YAP mRNA (e.g., NM_001130145 for human YAP1-2γ isoform) or TAZ mRNA (e.g., NM_000116 for human TAZ isoform-1).

MiRNAs for YAP or TAZ can be searched using the same databases as those described for miRNAs for TEAD3. For example, according to TarBase (see above), miRNAs for YAP1 include, but are not limited to, hsa-miR-204-5p and hsa-miR-506-3p. MiRNAs for TAZ include, but are not limited to, hsa-miR-382-3p and hsa-miR-26b-5p. Sequence information for these miRNAs and/or pre-miRNAs can be obtained, for example, using miRBase (see above).

Antisense nucleic acids and ribozyme nucleic acids for YAP and TAZ can also be designed and used in the same manner as the antisense nucleic acids and ribozyme nucleic acids for TEAD3 described above.

In another embodiment, 14-3-3 proteins can be used as inhibitors of YAP or TAZ. Enriching intracellular 14-3-3 proteins can promote the formation of complexes with YAP/TAZ and inhibit the nuclear translocation of YAP/TAZ.
In another embodiment, activators of the Hippo signaling pathway can also be used as inhibitors of YAP or TAZ. Activation of the Hippo signaling pathway promotes phosphorylation of YAP/TAZ, suppressing their nuclear translocation. Examples of activators of the Hippo signaling pathway include Lats1/2 (and their coactivators Mob1A/1B) and, further upstream, MST1/2 (and their coactivators WW45).
In yet another embodiment, dominant-negative mutants of YAP or TAZ can be used. The TEAD-binding domain of YAP and TAZ is 50-100 amino acids and 13-57 amino acids from the N-terminus, respectively, and the transcriptional activation domain is 276-472 and 208-381, respectively. Therefore, when translocated into the nucleus, a YAP or TAZ fragment containing the TEAD-binding domain but lacking the transcriptional activation domain can competitively bind to TEAD3 with endogenous YAP/TAZ and suppress the transcriptional activation of target genes mediated by the interaction between TEAD3 and YAP/TAZ. If nuclear localization is impaired due to deletion of the transcriptional activation domain, a known nuclear localization signal sequence may be added.
The amino acid sequences and mRNA sequences of these YAP/TAZ inhibitors are all known, and sequence information is available from various databases. The desired proteins or nucleic acids encoding them can be obtained in the same manner as for the above-described dominant-negative mutants of TEAD3 and the nucleic acids encoding them. The obtained proteins or nucleic acids can be introduced into somatic cells in the same manner as for the dominant-negative mutants of TEAD3 or the nucleic acids encoding them.

(h) Decoy nucleic acid for TEAD3
YAP/TAZ conjugates not only with TEAD3 but also with other transcription factors. Therefore, to selectively suppress the transcriptional activation of target genes using TEAD3, it is preferable to use a substance that inhibits the binding of TEAD3 to the promoter region of the target gene. Such substances include the above-mentioned dominant-negative mutants of TEAD3 lacking the YAP/TAZ-binding domain (transcriptional activation domain), as well as decoy nucleic acids containing the TED3 consensus binding sequence. An example of the TEAD3 consensus binding sequence is ACATTCCA. Preferably, the decoy nucleic acid is double-stranded DNA. The length of the decoy nucleic acid is, for example, 8 to 30 nucleotides, preferably 8 to 20 nucleotides.

Decoy nucleic acids for TEAD3 can be prepared in the same manner as the oligonucleic acids containing the consensus binding sequence for p53 described above and introduced into somatic cells.

The above-mentioned TEAD3 function inhibitor must be introduced into somatic cells in a manner sufficient to inhibit TEAD3 function during the nuclear reprogramming process of somatic cells. Here, nuclear reprogramming of somatic cells can be carried out by introducing a nuclear reprogramming substance into the somatic cells.

(C) Nuclear reprogramming substance. In the present invention, a "nuclear reprogramming substance" refers to any substance or substances that can induce iPS cells from somatic cells by introducing them into the somatic cells, and may be composed of any substance, such as a proteinaceous factor or a nucleic acid encoding the same (including in a form incorporated into a vector), or a low-molecular-weight compound. When the nuclear reprogramming substance is a proteinaceous factor or a nucleic acid encoding the same, preferred examples include the following combinations (hereinafter, only the names of the proteinaceous factors are given):
(1) Oct3/4, Klf4, c-Myc
(2) Oct3/4, Klf4, c-Myc, Sox2 (wherein Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18, preferably Sox1, Sox3, Sox15, or Sox17, more preferably Sox1 or Sox3. Klf4 can be replaced with Klf1, Klf2, or Klf5, preferably Klf2. c-Myc can be replaced with T58A (active mutant), N-Myc, or L-Myc.)
(3) Oct3/4, Klf4, c-Myc, Sox2, Fbx15, Nanog, Eras, ECAT15-2, TclI, β-catenin (active mutant S33Y)
(4) Oct3/4, Klf4, c-Myc, Sox2, TERT, SV40 Large T antigen (hereinafter SV40LT)
(5) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E6
(6) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E7
(7) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV6 E6, HPV16 E7
(8) Oct3/4, Klf4, c-Myc, Sox2, TERT, Bmil
(For the above, see WO 2007/069666 (however, for the substitution of Sox2 with Sox18 and the substitution of Klf4 with Klf1 or Klf5 in the combination (2) above, see Nature Biotechnology, 26, 101-106 (2008)). For the combination of "Oct3/4, Klf4, c-Myc, Sox2", see also Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007). For the combination of "Oct3/4, Klf2 (or Klf5), c-Myc, Sox2", see also Nat. Cell Biol., 11, 197-203 (2009). For the combination of "Oct3/4, Klf4, c-Myc, Sox2, hTERT, For the combination of "SV40LT", see Nature, 451, 141-146 (2008).
(9) Oct3/4, Klf4, Sox2 (see Nature Biotechnology, 26, 101-106 (2008)) (Here, Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18. Also, Klf4 can be replaced with Klf1, Klf2, or Klf5.)
(10) Oct3/4, Sox2, Nanog, Lin28 (Science, 318, 1917-1920 (2007))
(11) Oct3/4, Sox2, Nanog, Lin28, hTERT, SV40LT (See Stem Cells, 26, 1998-2005 (2008))
(12) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28 (see Cell Research (2008) 600-603)
(13) Oct3/4, Klf4, c-Myc, Sox2, SV40LT (see also Stem Cells, 26, 1998-2005 (2008))
(14) Oct3/4, Klf4 (see Nature 454:646-650 (2008), Cell Stem Cell 2:525-528 (2008))
(15) Oct3/4, c-Myc (see Nature 454:646-650 (2008))
(16) Oct3/4, Sox2 (Nature, 451, 141-146 (2008), see WO2008/118820)
(17) Oct3/4, Sox2, Nanog (see WO2008/118820)
(18) Oct3/4, Sox2, Lin28 (see WO2008/118820)
(19) Oct3/4, Sox2, c-Myc, Esrrb (where Esrrb can be replaced by Esrrg. See Nat. Cell Biol., 11, 197-203 (2009))
(20) Oct3/4, Sox2, Esrrb (see Nat. Cell Biol., 11, 197-203 (2009))
(21) Oct3/4, Klf4, L-Myc
(22) Oct3/4, Nanog
(23) Oct3/4 (Cell 136: 411-419 (2009), Nature, 08436, doi:10.1038 published online(2009))
(24) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28, SV40LT (Science, 324: 797-801 (2009))
(25) Oct3/4, Sox2, Klf4, L-Myc, Lin28
(26) Oct3/4, Sox2, Klf4, L-Myc, Lin28, Glis1

In the above (1)-(26), other Oct family members, such as Oct1A and Oct6, can be used in place of Oct3/4. Furthermore, other Sox family members, such as Sox7, can be used in place of Sox2 (or Sox1, Sox3, Sox15, Sox17, and Sox18). Furthermore, combinations that do not fall under the above (1)-(26) but contain all of the components of any of them and further contain any other substances can also be included in the category of "nuclear reprogramming substances" in the present invention. Furthermore, under conditions where the somatic cells to be subjected to nuclear reprogramming endogenously express some of the components of any of the above (1)-(26) at levels sufficient for nuclear reprogramming, combinations of only the remaining components excluding those components can also be included in the category of "nuclear reprogramming substances" in the present invention.

Among these combinations, preferred examples of nuclear reprogramming substances include at least one, preferably two or more, and more preferably three or more selected from Oct3/4, Sox2, Klf4, c-Myc or L-Myc, Nanog, Lin28, and SV40LT.

In particular, when considering therapeutic use of the resulting iPS cells, a combination of the three factors Oct3/4, Sox2, and Klf4 (i.e., (9) above) is preferred. On the other hand, when therapeutic use of iPS cells is not considered (for example, when using them as research tools for drug discovery screening, etc.), an example of a combination of four factors, Oct3/4, Sox2, and Klf4 plus c-Myc, can be used. Alternatively, regardless of the manner in which the iPS cells will be used, examples of a combination of five factors, Oct3/4, Sox2, and Klf4 plus L-Myc and Lin28 (i.e., (25) above), or six factors, further including Glis1 (i.e., (26) above) and SV40 Large T, can be used, for example.

Furthermore, a combination in which c-Myc is replaced with L-Myc in the above is also an example of a preferred nuclear reprogramming substance.

The nucleotide sequences of the mouse and human cDNAs for each of the above nuclear reprogramming substances and the amino acid sequence information of the proteins encoded by the cDNAs can be obtained by referring to the NCBI accession numbers listed in WO 2007/069666, and the mouse and human cDNA and amino acid sequence information for L-Myc, Lin28, Lin28b, Esrrb, Esrrg, and Glis1 can be obtained by referring to the NCBI accession numbers listed below. Those skilled in the art can prepare the desired nuclear reprogramming substances using standard methods based on the cDNA sequences or amino acid sequence information.
Gene Name Mouse Human
L-Myc NM_008506 NM_001033081
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM_001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438
Glis1 NM_147221 NM_147193

When using a protein factor itself as the nuclear reprogramming substance, the resulting cDNA can be inserted into an appropriate expression vector and introduced into host cells. The cells can then be cultured and the recombinant protein factor recovered from the resulting culture. When using a nucleic acid encoding a protein factor as the nuclear reprogramming substance, the resulting cDNA can be inserted into a viral, episomal, or plasmid vector to construct an expression vector, as in the case of the nucleic acid encoding the dominant-negative mutant of TEAD3 described above, and then subjected to the nuclear reprogramming process. If necessary, the Cre-loxP system or piggyBac transposon system described above can also be used. When introducing nucleic acids encoding two or more protein factors as nuclear reprogramming substances into cells, each nucleic acid can be carried on a separate vector, or multiple nucleic acids can be linked in tandem to form a polycistronic vector. In the latter case, to enable efficient polycistronic expression, it is preferable to use a 2A sequence, such as the 2A sequence of foot-and-mouth disease virus (PLoS ONE 3, e2532, 2008; Stem Cells 25, 1707, 2007) or an IRES sequence (U.S. Patent No. 4,937,190).

Contact of a nuclear reprogramming substance with a somatic cell can be carried out (a) in the case where the substance is a protein factor, in the same manner as for the dominant-negative mutant of TEAD3 described above; or (b) in the case where the substance is a nucleic acid encoding the protein factor of (a), in the same manner as for the nucleic acid encoding the dominant-negative mutant of TEAD3 described above.

(D) Substances for improving iPS cell establishment efficiency In addition to the above-mentioned TEAD3 function inhibitors, contacting somatic cells with other known substances for improving establishment efficiency is expected to further increase the efficiency of iPS cell establishment.

Substances for improving the efficiency of iPS cell establishment include, for example, histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA) (Nat. Biotechnol., 26(7): 795-797 (2008)), trichostatin A, sodium butyrate, MC 1293, and M344, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool™ (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene))], DNA methyltransferase inhibitors (e.g., 5′-azacytidine) (Nat. Biotechnol., 26(7): 795-797 (2008)), and G9a histone methyltransferase inhibitors [e.g., BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), small molecule inhibitors such as siRNA and shRNA against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology)), nucleic acid expression inhibitors such as L-channel calcium agonists (e.g., Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), UTF1 (Cell Stem Cell, 3, 475-479 (2008)), Wnt signaling activators (e.g., soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 2i/LIF (2i is an inhibitor of mitogen-activated protein kinase signaling and glycogen synthase kinase-3, PloS Biology, 6(10), 2237-2247 (2008)), ES cell-specific miRNAs (e.g., miR-302-367 cluster (Mol. Cell. Biol. doi:10.1128/MCB.00398-08), miR-302 (RNA (2008) 14: 1-10), miR-291-3p, miR-294, and miR-295 (all of which are published in Nat. Biotechnol. 27: 459-461 (2009)), 3'-phosphoinositide-dependent kinase-1 (PDK1) acivator (e.g., PS48 (Cell Stem Cell, 7: 651-655 (2010))), neuropeptide Y (WO 2010/147395), prostaglandins (e.g., prostaglandin E2 and prostaglandin J2) (WO 2010/068955), and the like, but are not limited thereto.
The nucleic acid expression inhibitor may be in the form of an expression vector containing DNA encoding siRNA or shRNA.

Of the components of the nuclear reprogramming substances, for example, SV40 large T, which is a supplementary factor but not essential for the nuclear reprogramming of somatic cells, can also be included in the category of iPS cell establishment efficiency improvers. Given that the mechanism of nuclear reprogramming is currently unclear, it may be expedient to classify supplementary factors other than those essential for nuclear reprogramming as either nuclear reprogramming substances or iPS cell establishment efficiency improvers. In other words, because the somatic cell nuclear reprogramming process can be viewed as a holistic event that occurs upon contact of somatic cells with nuclear reprogramming substances and iPS cell establishment efficiency improvers, those skilled in the art will not necessarily need to clearly distinguish between the two.

These other substances that improve iPS cell establishment efficiency can be contacted with somatic cells using methods similar to those described above for TEAD3 function inhibitors, depending on whether the substance is (a) a proteinaceous factor or (b) a nucleic acid that encodes the proteinaceous factor. If the substance is a low-molecular-weight compound, it can be added to the culture medium for somatic cells at an appropriate concentration.

(E) Improving iPS cell establishment efficiency by culturing cells under hypoxic conditions during the nuclear reprogramming step of somatic cells can further improve iPS cell establishment efficiency. As used herein, "hypoxic conditions" refers to conditions in which the oxygen concentration in the atmosphere during cell culture is significantly lower than that in air. Specifically, these conditions include conditions with an oxygen concentration lower than the oxygen concentration in the 5-10% _CO /95-90% air atmosphere commonly used in conventional cell culture, such as conditions with an oxygen concentration of 18% or less. Preferably, the oxygen concentration in the atmosphere is 15% or less (e.g., 14% or less, 13% or less, 12% or less, 11% or less, etc.), 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less, etc.), or 5% or less (e.g., 4% or less, 3% or less, 2% or less, etc.). The oxygen concentration in the atmosphere is preferably 0.1% or more (e.g., 0.2% or more, 0.3% or more, 0.4% or more, etc.), 0.5% or more (e.g., 0.6% or more, 0.7% or more, 0.8% or more, 0.95% or more, etc.), or 1% or more (e.g., 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more, etc.).
For more detailed culture conditions regarding hypoxic culture, see, for example, WO 2010/013845.

After contacting the cells with the nuclear reprogramming substance and the TEAD3 function inhibitor, the cells can be cultured under conditions suitable for culturing ES cells, for example. Mouse cells are cultured in standard culture medium supplemented with leukemia inhibitory factor (LIF) as a differentiation inhibitor. Human cells, on the other hand, are preferably cultured with basic fibroblast growth factor (bFGF) and/or stem cell factor (SCF) instead of LIF. Furthermore, cells are typically cultured in the presence of mouse embryonic fibroblasts (MEFs) that have been treated with radiation or antibiotics to arrest cell division. While STO cells are commonly used as MEFs, SNL cells (McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990)) are commonly used to induce iPS cells. Co-culture with feeder cells can be initiated before, at the time of, or after contact (e.g., 1-10 days after contact).

Candidate iPS cell colonies can be selected using drug resistance and reporter activity as indicators, or by visual morphological observation. The former involves using recombinant cells in which a drug resistance gene and/or reporter gene is targeted to the locus of a gene that is specifically highly expressed in pluripotent cells (e.g., Fbx15, Nanog, Oct3/4, etc., preferably Nanog or Oct3/4), and selecting colonies that are positive for drug resistance and/or reporter activity. Examples of such recombinant cells include MEFs derived from mice in which the βgeo gene (encoding a fusion protein of β-galactosidase and neomycin phosphotransferase) was knocked into the Fbx15 locus (Takahashi & Yamanaka, Cell, 126, 663-676 (2006)), and MEFs derived from transgenic mice in which the green fluorescent protein (GFP) gene and puromycin resistance gene were integrated into the Nanog locus (Okita et al., Nature, 448, 313-317 (2007)). On the other hand, methods for selecting candidate colonies by visual morphological observation include the method described in Takahashi et al., Cell, 131, 861-872 (2007). While methods using reporter cells are simple and efficient, visual colony selection is preferable for generating iPS cells for human therapeutic use from the perspective of safety.

The identity of the cells in the selected colonies as iPS cells can be confirmed by the above-mentioned Nanog (or Oct3/4) reporter positivity (puromycin resistance, GFP positivity, etc.) and visual inspection of the formation of ES cell-like colonies. However, for greater accuracy, tests such as alkaline phosphatase staining, analysis of the expression of various ES cell-specific genes, or transplantation of the selected cells into mice to confirm teratoma formation can also be performed.

iPS cells established in this way can be used for a variety of purposes. For example, differentiation methods reported for ES cells can be used to induce differentiation of iPS cells into various cell types (e.g., cardiomyocytes, blood cells, neurons, vascular endothelial cells, insulin-secreting cells, etc.). Therefore, if iPS cells are derived from somatic cells collected from the patient or from a donor with the same or substantially identical HLA type, they can be differentiated into desired cells (i.e., cells of the patient's affected organ or cells that have a therapeutic effect against the disease) and transplanted into the patient, enabling autologous stem cell therapy. Furthermore, functional cells (e.g., hepatocytes) differentiated from iPS cells are thought to more closely reflect the actual state of these cells in vivo than corresponding existing cell lines, and therefore can be suitably used for in vitro screening of the efficacy and toxicity of drug candidate compounds.

The present invention will be further explained below with reference to examples, but the present invention is not limited to the following examples in any way.

Materials and Methods [Method 1] Cell Culture. Primary culture of mouse embryonic fibroblasts (MEFs) was performed according to a previously described established method (Okita et al., 2007). MEFs were cultured in Dulbecco's modified Eagle's medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37°C under 5% _CO2 . DMEM was supplied with 0.5% penicillin and streptomycin (Invitrogen). Human dermal fibroblasts (HDFs) were cultured under similar conditions. MEF- and HDF-derived iPS cells were cultured in DMEM supplemented with leukemia inhibitory factor (LIF), containing 15% FBS, 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), and 0.5% penicillin and streptomycin.

[Method 2] Preparation of mouse iPS cells
iPS cells were established as previously described (Okita et al., 2007; Takahashi and Yamanaka, 2006) with some modifications. MEFs were seeded at 1 × ¹⁰ cells per well and cultured overnight. After 24 hours, the MEFs were transduced with four factors (Oct3/4, Sox2, Klf4, and c-Myc; sometimes abbreviated as "4F" herein) by infection with a retrovirus containing a Nanog-GFP cassette. After 24 hours, the cells were passaged once and seeded at 2.5 × ¹⁰ cells per dish onto a feeder layer of mitomycin C-treated SNL cells. The next day, the medium was replaced with mouse iPS cell medium and cultured for 30 days.

[Method 3] Generation of human iPS cells using retroviral vectors
iPS cells were established as previously described (Takahashi et al., 2007) with minor modifications. HDFs were seeded at 1 × ¹⁰ cells per well and cultured overnight. After 24 hours, four factors (4F: OCT3/4, SOX2, KLF4, and c-MYC) or three factors (4F minus c-MYC, sometimes referred to as "3F" in this paper) were introduced into the HDFs by retroviral infection. After 96 hours, the cells were passaged and seeded onto a feeder layer of mitomycin C-treated SNL cells at 2.5 × ¹⁰ cells (4 factors) or 5 × ¹⁰ cells (3 factors). The next day, the medium was replaced with primate ES medium (ReproCELL, Japan) supplemented with 4 ng/mL human basic fibroblast growth factor (bFGF) and then cultured for 30 days.

[Method 4] Generation of human iPS cells using episomal vectors
iPS cells were established as previously described (Okita et al., 2011) with minor modifications. HDFs were cultured in DMEM supplemented with 10% FBS. According to the Neon transfection system (Invitrogen) instructions, 3 μg of the expression plasmid mixture (pCXLE-hOCT3/4-shp53, pCXLE-hSOX2-hKLF4, and pCXLE-hLIN28-hL-MYC) was electroporated into 6 × 10 ⁵ HDFs using 100 μL of the kit solution. Electroporation was performed three times (3 pulses) at 1650 V for 10 ms. Four days after transduction, the cells were trypsinized and replated at a density of 2 × 10 ⁵ cells onto a 100 mm dish covered with a feeder layer of mitomycin C-treated SNL cells. The next day, the medium was replaced with primate ES cell medium supplemented with bFGF, and the cells were then cultured for 30 days.

[Method 5] Alkaline Phosphatase Staining and Immunocytochemistry Alkaline phosphatase (AP) staining was performed according to the protocol of the alkaline phosphatase detection kit (Sigma). For immunocytochemical analysis, the cells were fixed in 4% paraformaldehyde in PBS at room temperature for 20 minutes. After washing with PBS, the cells were treated with blocking solution (PBS containing 5% normal goat serum (Millipore), 1% bovine serum albumin (BSA, Nacalai Tesque), and 0.2% Triton X-100) at room temperature for 45 minutes. The primary antibodies and dilutions were as follows:
Antibodies used were anti-OCT4 (1:50, Santa Cruz, sc-5279), anti-SOX2 (1:100, Abcam, ab75485), and anti-TRA1-60 (1:50, Millipore, MAB4360). Alexa Fluor 488-conjugated anti-mouse IgG (1:500, Invitrogen, A-11001) was used as the secondary antibody. Hoechst 33342 (1 μg/mL, Invitrogen) was used for nuclear staining.

[Method 6] Differentiation induction
iPS cells were harvested using CTK solution containing 0.25% trypsin (Invitrogen), 0.1 mg/mL collagenase IV (Invitrogen), 20% KSR, and 0.1 mM _CaCl2 . The resulting cell clusters were suspended in primate ES medium containing 10 μM Y-27632 (Wako) without bFGF and seeded onto ultra-low binding plates (Corning) to form embryoid bodies. The embryoid bodies were cultured in suspension for 8 days, then seeded onto gelatin-coated plates and cultured for an additional 8 days. The cells were then subjected to immunocytochemical analysis. The primary antibodies used were as follows:
Anti-Tuj1 antibody (1:100, Chemicon: MAB1637), anti-α-smooth muscle actin antibody (α-SMA, 1:500, DAKO: M085101), and anti-α-fetoprotein antibody (1:100, R&D: MAB1368) were used. Alexa488-conjugated anti-mouse IgG (1:500, Invitrogen: A-11001) was used as the secondary antibody.

[Method 7] Microarray Preprocessing and Differential Gene Expression Analysis. Microarray slides were scanned using a single-color Agilent DNA microarray scanner and analyzed using default parameters. Raw data were loaded into RStudio (R visual scripting), and gene expression data for reading, searching, and preprocessing were analyzed using the Bioconductor package Limma. The Limma workflow was used for differential gene expression analysis (DEGs). Hierarchical cluster derived trees were constructed using hclust from the stat package and agnes from the cluster package. Distances were calculated using the Manhattan city-block distance method, and k-means were calculated using the kmeans function. Distance and correlation matrices were visualized using get_dist and fviz_dist from the factoroextra package. Cluster scatter plots were calculated using the fviz_cluster function. Expression data for DEGs were clustered using R scripts, and heatmaps were generated. Gene ontology and gene cluster statistical analysis were performed using the DOSE and clusterProfiler packages. The microarray data obtained in this study are available in the Gene Expression Omnibus under the accession number GSE56167.

[Method 8] Gene silencing
Continuous knockdown of the p53 gene was achieved using short hairpin RNA (shRNA) as previously described (Hong et al., 2009; Masutomi et al., 2003) with minor modifications. The shRNA against the p53 gene (pMKO.1-puro p53 shRNA-2; Addgene plasmid 10672) or a mock vector (pMXs) was introduced into MEFs or HDFs by retroviral infection along with 4F.
Continuous knockdown of the TEAD3 gene was achieved using two shRNAs, sh#1 and sh#2, directed against TEAD3 mRNA (target mRNA sequences: sh#1 5'-AGCATGACCATCAGCGTCTCCACCAAGGT-3' (SEQ ID NO: 5); sh#2 5'-AGCAACCAGCACAATAGCGTCCAACAGCT-3' (SEQ ID NO: 4)). HDFs were seeded at 1 × ¹⁰ cells/well in a 6-well plate and cultured overnight. The next day, shRNA or a mock vector (pSINsi-hH1) was retrovirally transfected into the HDFs along with 4F. After 4 days, the cells were harvested by trypsinization and seeded at 2 × ¹⁰ cells/100 mm dish onto a mitomycin C-treated SNL cell feeder layer.

[Method 9] Teratoma formation
iPS cells were harvested using CTK solution and seeded in 60 mm dishes. After culturing until confluent, the cells were harvested and injected into the testes of non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice (CREA, Japan). Three months after injection, the resulting tumors were dissected and fixed in 4% paraformaldehyde in PBS. Thin sections from the paraffin-embedded tissue were stained with hematoxylin and eosin.

Animal welfare This study was carried out in strict compliance with the recommendations of the Kyoto University Animal Experiment Regulations.

result
1) The inhibitory effect of p38 on the reprogramming of mouse embryonic fibroblasts (MEFs)
iPSCs were generated from MEFs according to Method 2. At 24 hours after retroviral transduction of 4F, DMSO (vehicle, negative control), a p38-selective inhibitor (SB202190, Calbiochem, 10 μM), or one of seven compounds known to promote MEF reprogramming efficiency was added to the culture medium. After 98 hours, the medium was replaced with a medium containing no DMSO or p38-selective inhibitor. The seven compounds and their concentrations were as follows: vitamin C (Sigma, 10 μg/mL), valproic acid (Sigma, 1.9 mM), CHIR99021 (Calbiochem, 3 μM), PD0325901 (Calbiochem, 0.5 μM), interleukin-6 (R&D, 0.2 ng/mL), AS601245 (Calbiochem, 5 μM), and rapamycin (Sigma, 1 μM). As a negative control for 4F transduction, cells infected with a retrovirus (empty vector) encoding no 4F were treated with the same drugs. To evaluate the efficiency of viral infection, the cells were infected with a retrovirus into which the 4F and DsRed genes had been inserted (4F+DsRed), and the same procedure was carried out.

The number of GFP-positive colonies was counted 21 days (Day 21) and 28 days (Day 28) after 4F transduction and compared with the number of GFP-positive colonies in the 4F-transduced negative control to calculate the reprogramming efficiency. Correction was performed using the number of GFP-positive colonies obtained by infection with the retrovirus containing the 4F and DsRed genes (4F+DsRed) (correction for viral infection efficiency). The results are shown in Figure 1A. Similar corrections were also performed in the subsequent analysis of reprogramming efficiency, but a detailed explanation is omitted here.
As shown in Figure 1A, MEFs treated with the p38-selective inhibitor (SB202190) yielded nearly twice as many GFP-positive colonies as MEFs treated with DMSO on both Day 21 and Day 28. The number of GFP-positive colonies was almost equivalent to that obtained with treatment with the antioxidant (VC, vitamin C) and GSK3β inhibitor (CH, CHIR99021), which were the most effective of the seven known compounds.

Next, analysis was performed by varying the duration of drug treatment. DMSO or SB202190 was added to the medium for four periods (A: Days 1-4, B: Days 1-8, C: Days 8-16, D: Days 1-16) as shown in Figure 1B, and the number of GFP-positive colonies was counted on Days 21 and 28. The results are shown in Figure 1C.
As shown in Figure 1C, the experimental group treated with SB202190 during the early phase (Period A) showed a significant increase in the number of GFP-positive colonies at both Day 21 and Day 28 compared with the control (DMSO-treated during the same period). Furthermore, as shown in Figure 1D, the experimental group treated with SB202190 during Period A tended to have a higher number of GFP-positive colonies on Day 28 than on Day 21. Furthermore, as shown in Figure 1E, the experimental group treated with SB202190 during Period A had significantly higher numbers of GFP-positive colonies at both Day 21 and Day 28 than the experimental group treated with SB202190 during Period D. Interestingly, long-term treatment with SB202190 suppressed iPS cell proliferation (Figure 7A), suggesting that p38 inhibition during the late phase may impair reprogramming. Chimeric embryos were generated from mouse iPS cells obtained by treatment with SB202190 during the early phase (period A) and transplanted into foster mothers. The embryos successfully differentiated into multiple tissues (Figure 7B), and germline transmission was also confirmed (Figure 7C).

These results demonstrate that inhibiting p38 during reprogramming of mouse cells by expressing reprogramming factors significantly increases the reprogramming efficiency. Furthermore, it was revealed that inhibiting p38 during the early phase of reprogramming in mouse cells has a very significant effect in promoting reprogramming.

2) The inhibitory effect of p38 on the reprogramming of human dermal fibroblasts (HDFs)
We investigated whether p38 inhibition has a similar effect on human cells using several p38-selective inhibitors. During the process of generating iPSCs from HDFs (Method 3), DMSO (negative control) or one of the p38-selective inhibitors (SB202190, SB203580, or SB239063 (all Calbiochem, final concentration 10 μM)) was added to the culture medium at four different time periods (A–D) as shown in Figure 2A. GFP-positive colonies were counted 16 days (Day 16), 24 days (Day 24), and 32 days (Day 32) after 4F transduction. The reprogramming efficiency was calculated according to the method for calculating MEF reprogramming efficiency (see section 1) above. Figure 2B shows the results for the experimental groups treated with the inhibitors at time periods A and D. In the experimental groups treated with inhibitors during Period A, the number of GFP-positive colonies on Day 24 and Day 32 was significantly higher than that of the control (DMSO-treated) experimental group, regardless of the type of inhibitor used (Fig. 2B, left bar). Furthermore, in the experimental groups treated with inhibitors during Period D, the number of GFP-positive colonies tended to be significantly higher than that of the control, regardless of the type of inhibitor used (Fig. 2B, right bar). In particular, when SB202190 or SB203580 was added throughout the entire phase (Period D), the number of GFP-positive colonies on Day 32 was significantly increased by more than two-fold compared to when SB202190 or SB203580 was added only during the initial phase.

These results demonstrate that inhibiting p38 during the reprogramming process significantly increases the reprogramming efficiency in both mouse and human cells. Furthermore, in human cell reprogramming, inhibiting p38 throughout the entire reprogramming phase, not just the initial phase, was highly effective in promoting reprogramming (more than twice as effective as inhibiting p38 only during the reprogramming phase).
Furthermore, the above results suggest that there may be differences in the role of p38 in the reprogramming process between mouse and human cells, with p38 more strongly inhibiting reprogramming in human cells.

Human iPSCs obtained by treatment with SB202190 maintained an ESC-like morphology even after 30 passages from Day 32 (Figure 2C) and expressed the ESC-specific marker alkaline phosphatase (Figure 2D, [Method 5]). Furthermore, expression of OCT4, SOX2, and TRA-1-60 was observed (Figure 2E, [Method 5]), confirming their human ESC-like marker profile.

Furthermore, a similar analysis was performed when reprogramming was performed by introducing and expressing 3F (Oct3/4, Sox2, and Klf4) instead of 4F using a retroviral vector, or 4F using an episomal vector. Cells were treated with SB202190 during Period A, and the number of GFP-positive colonies was counted on Day 24 and Day 32. The results are shown in Figure 2F and G.

When cells were reprogrammed with 3F (Figure 2F) or when 4F was expressed using an episomal vector (Figure 2G), the number of GFP-positive colonies ultimately obtained was significantly increased compared to the control (DMSO) group.

These results demonstrate that inhibiting p38 significantly increases the reprogramming efficiency of human cells, regardless of the type of reprogramming factor or the method of reprogramming factor introduction. Furthermore, treating HDFs or 4F-introduced HDFs with a p38 inhibitor for 96 hours promoted cell proliferation (Figures 8A and B). However, similar treatment of HDF-derived iPSCs did not alter the proliferation rate of human iPSCs (Figure 8C). These results suggest that p38 inhibition does not increase iPSC numbers by promoting human iPSC proliferation.

3) Differentiation Potential of Human iPSCs Obtained by Inhibiting p38 We analyzed the differentiation potential of human iPSCs obtained by inhibiting p38 during reprogramming. Three clones (SB1-SB3) were established from HDF-derived iPSCs obtained by SB202190 treatment, and the expression levels of SOX2, OCT4, and NANOG, indicators of pluripotency, were analyzed for these clones (Figure 3A). As shown in Figure 3A, SB1-SB3 showed increased mRNA levels of SOX2, OCT4, and NANOG compared to HDFs (HD), comparable to or greater than those of human iPSCs (DM, B7) and embryonic stem cells (ES) obtained by reprogramming without inhibiting p38. Karyotyping confirmed normal karyotypes (Figure 3B), indicating that p38 inhibition during reprogramming does not impair chromosomal stability. Furthermore, when these clones were induced to differentiate into three germ layers via embryoid bodies (Method 6), they differentiated into mesoderm expressing smooth muscle actin (A-SMA), ectoderm expressing beta-III tubulin (B-3-TUBULIN), and endoderm expressing alpha-fetoprotein (AFP) (Figure 3C). Furthermore, when their in vivo teratoma formation ability was analyzed according to Method 9, teratomas were formed from all clones analyzed, confirming that they differentiated into three germ layers, including neuroepithelium, cartilage, and various glandular structures (Figure 3D). Therefore, human iPSCs obtained by inhibiting p38 during reprogramming possess the ability to differentiate into three germ layers both in vitro and in vivo.

We further analyzed the effect of p38 inhibition on the reprogramming efficiency of HDFs derived from four new donors (HDF1616, HDF1079, HDF1078, and Tig109). As in 2) above, HDFs were treated with SB202190 during period D of the reprogramming process, and the number of GFP-positive colonies was counted on Day 32. The results are shown in Figure 3E. Regardless of the donor-derived HDFs used, the number of GFP-positive colonies was significantly increased in the SB202190-treated experimental group compared to the control, confirming that p38 inhibitor treatment significantly promoted reprogramming efficiency.

These results confirm that human iPSCs obtained by inhibiting p38 during the reprogramming process are comparable to human iPS cells and ES cells obtained without p38 inhibition, have a normal karyotype, and are capable of differentiating into the three germ layers.

4) Analysis of genes whose expression levels are reduced by both p38 and p53
p53 is a gene that functions as a powerful reprogramming barrier during reprogramming of human and mouse cells. As shown in Figure 4B, continuous knockdown of p53 using shRNA against p53 mRNA (Figure 4A) during reprogramming with 4F (Method 8) significantly increased the number of iPS cell colonies (shp53 bar). Surprisingly, in the experimental group where continuous p53 knockdown was combined with SB202190 treatment during Period A (shp53 + SB202190 bar), the number of iPS cell colonies obtained was significantly greater than the sum of the increases in iPS cell colonies obtained by each treatment alone (shp53, SB202190 bar) compared to the control (DMSO bar) (Figure 4B). Therefore, p53 knockdown and p38 inhibition demonstrated a synergistic (promoting) effect on reprogramming. These results suggest the existence of a transcription factor commonly regulated, either directly or indirectly, by p53 and p38.

To investigate the relationship between changes induced by p53 knockdown and p38 inhibition and their correlation with increased reprogramming efficiency (ΔiPSC colony count), we performed principal component analysis (PCA) based on the total mRNA transcriptome. Three principal components (PC1 54.37%, PC2 19.64%, PC3 18.57%) were identified, and the expression levels of these components were plotted for each sample to confirm changes in expression profile (Figure 4C). Furthermore, hierarchical clustering analysis of these pluripotent transcriptomes classified them into three clusters (Figure 4D, upper panel). SB202190 treatment and shp53/SB202190 double treatment were classified into the same cluster (Cluster C), suggesting similarities in certain components. However, sample correlation matrix analysis indicated that many genes were differentially regulated by the four treatments, with the largest differences observed between DMSO treatment and double treatment (Figure 4D, lower panel). Similarly, the k-means algorithm on the cluster scatter plot showed that cluster A (DMSO-treated group) was mapped most distally from the other clusters, indicating that the pluripotency transcriptomes regulated by either shp53 or p38 inhibition were most separated along the first dimension (Fig. 4E), consistent with the PCA analysis described above.

To identify key factors for increased reprogramming efficiency, we performed differentially expressed gene (DEG) analysis between each cluster. As a result, we found 1,147 DEGs induced by SB202190 and 2,185 DEGs induced by shp53 (Figure 4F), revealing significant changes in gene expression between each cluster.

5) Identification of genes that function as reprogramming barriers. To identify genes that function as strong barriers to reprogramming, we first analyzed genes whose expression levels were reduced between the shp53-treated group and the shp53/SB202190 double-treated group. Of these, 651 genes were identified as genes whose expression levels were specifically reduced in the double-treated group (DOWN1 in Figure 5A). Similarly, we analyzed genes whose expression levels were reduced between the SB202190-treated group and the shp53/SB202190 double-treated group. Of these, 1,056 genes were identified as genes whose expression levels were specifically reduced in the double-treated group (DOWN2 in Figure 5B). Furthermore, by using a 2-fold cutoff to exclude genes with a fold change of 2 or less in the analyses of the shp53-treated group and the SB202190-treated group and the double-treated group, we identified 340 genes that were common to both DOWN1 and DOWN2 (Figure 5C).

Of the 340 genes, 31 were classified as transcription factors or genes with DNA binding activity (Figure 5D). GO enrichment analysis of the 340 genes revealed the following main GO terms: tyrosine kinase activity/membrane receptor kinase activity/membrane receptor tyrosine kinase activity, collagen binding/collagen receptor activity, and oligosaccharyltransferase activity (Figure 5E).

After extensive investigation of these 31 genes, the inventors ultimately identified TEAD3 as a gene that functions as a strong barrier to somatic cell reprogramming. First, they investigated the relationship between the effects of p53 and/or p38 inhibition on 4F-mediated HDF reprogramming and endogenous TEAD3 expression. Compared with 4F transfection alone (vehicle (DMSO) treatment), the single inhibition of p53 or p38 significantly reduced TEAD3 expression, whereas the dual inhibition of p53 and p38 significantly reduced TEAD3 expression (Figure 6A).

6) Increased reprogramming efficiency by inhibiting TEAD3 expression. Next, we analyzed the role of TEAD3 in reprogramming using two shRNAs specific to TEAD3 (4F-shTEAD3#1 and #2, Figure 6B). Expression of these shRNAs during 4F-mediated HDF reprogramming (Method 2) significantly reduced TEAD3 expression (Figure 6C) and significantly increased the number of GFP-positive colonies (Figure 6D). Furthermore, colonies obtained by TEAD3 shRNA expression were confirmed to be alkaline phosphatase-positive (Figure 6E). Therefore, reducing TEAD3 expression during HDF reprogramming significantly increased reprogramming efficiency.

We investigated the correlation between TEAD3 and p53 expression using the GSE36664 dataset (J Biol Chem 2012 Oct 19;287(43):35825-37), which contains transcriptome data from MEFs, the GSE45276 dataset (Mol Cell 2011 Apr 8;42(1):36-49), which contains transcriptome data from human lung fibroblasts, and the HDF transcriptome dataset. A positive correlation was observed in all cell types (Figure 9), suggesting that p53 positively regulates the expression of TEAD3, a reprogramming barrier. Analysis of the HDF single-cell RNA-Seq dataset revealed a positive correlation between TEAD3 and MAPK13 (p38δ), ERK4 (MAPK4), and p44-ERK1 (MAPK3), as well as a negative correlation between TEAD3 and cyclin-dependent kinases, such as CDK4 and CDK6, which regulate cell cycle progression (Figure 10).

To investigate whether the increased colony-forming ability of iPSCs through TEAD3 inhibition, via its contribution to cell cycle kinetics, is related to tumorigenesis, we examined the tumor-forming ability of HeLa cells with different levels of TEAD3 expression. The number and size of tumor-like nodules derived from cells in which TEAD3 expression was reduced by shRNA transfection were significantly increased compared with those derived from control cells (Figure 11). Furthermore, TEAD3 expression was reduced in human cervical cancer tumor tissue compared with normal tissue, confirming the role of TEAD3 in cancer progression (Figure 12).

HDFs transfected with shRNAs against 4F and TEAD3 showed a faster reprogramming rate than those transfected with 4F alone (4F + nonspecific shRNA) (Figure 13A and B). This may be partially explained by faster cell proliferation during the early phase of reprogramming (Figure 13C and E). However, once pluripotency was achieved, human iPSCs transfected with 4F alone and those transfected with shRNA against TEAD3 proliferated at similar rates (Figure 13D and F), suggesting the safety of iPSC clones derived by TEAD3 inhibition through restoration of cell cycle checkpoints.

Using the GSE116309 dataset (see Stem Cell Reports 2019 Feb 12;12(2):319-332), we examined the expression of TEAD3 during the reprogramming process from MEFs to iPS cells. TEAD3 expression was found to be rapidly downregulated immediately prior to the acquisition of pluripotency (Figure 6F). SSEA1 is an early reprogramming marker in MEFs, indicating successful commitment to reprogramming. Therefore, using the GSE106835 dataset (see Cell Stem Cell 2018 Aug 2;23(2):289-305.e5), we examined the expression of TEAD3 during the reprogramming process from MEFs to iPS cells in SSEA1-positive and SSEA1-negative cells, respectively. TEAD3 expression was downregulated only in SSEA1-positive MEF reprogramming intermediates upon acquisition of pluripotency (Figure 6G). This indicates that only cells that successfully commit to reprogramming require reduced TEAD3 expression to overcome cellular mechanisms that attempt to block reprogramming.

Furthermore, we explored the regulatory mechanism of TEAD3 expression. First, we obtained the sequence of the 5'-upstream promoter region from genomic DNA based on the TEAD3 mRNA sequence. Using known transcription start site (TSS) sequences as a query, we performed BLAST analysis against this promoter sequence and found a sequence that matched 100% with AGGGCGGAGC (SEQ ID NO: 6), a TSS consensus sequence. We then searched for transcription factors that could bind to this TSS sequence and predicted that KLF4 could bind to this TSS sequence (Figure 6H). Furthermore, analysis of the binding cis-element sequences of target genes known to be KLF4-bound identified the KLF4 binding consensus sequence GGGCGGGGC (SEQ ID NO: 7), which was highly homologous to the TEAD3 TSS sequence (Figure 6I). Since KLF4 has been suggested to be partially regulated by p38, inhibition of p38 may promote pluripotency by suppressing TEAD3 transcription.
Furthermore, a search for the promoter region of TEAD3 revealed the presence of a consensus sequence to which p53 can bind.

These results demonstrate that TEAD3 functions as a powerful barrier that prevents the reprogramming of human cells, and that inhibiting its expression or activity promotes the reprogramming efficiency of these cells.

According to the present invention, inhibiting TEAD3 function in the nuclear reprogramming process significantly improves iPS cell establishment efficiency, equivalent to dual inhibition of the p38 and p53 pathways. Because the method of the present invention is effective for reprogramming using a variety of methods, it is extremely useful in terms of both safety and cost for the application of human iPS cells to regenerative medicine.

This application is based on Patent Application No. 2020-101932 filed in Japan on June 11, 2020, the contents of which are incorporated herein by reference in their entirety.

Claims (5)

A method for improving the efficiency of establishing induced pluripotent stem (iPS) cells, comprising inhibiting the transcriptional activation function of transcriptional enhancer-associated domain family member-3 (TEAD3) in a group of genes that maintain somatic cell identity during the nuclear reprogramming process of somatic cells ,
The following (a) to (e):
(a) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the TEAD3 gene;
(b) an antisense nucleic acid against a transcript of the TEAD3 gene;
(c) a ribozyme nucleic acid against a transcription product of the TEAD3 gene;
(d) a dominant-negative mutant of TEAD3 or a nucleic acid encoding the same; or
(e) Decoy nucleic acid for TEAD3
A method for inhibiting the transcriptional activation function of a group of genes that maintain the somatic specificity of TEAD3 by introducing the gene into somatic cells . An agent for improving iPS cell establishment efficiency, comprising an inhibitor of the transcription activation function of a group of genes that maintain the somatic identity of TEAD3,
The inhibitor is one of the following (a) to (e):
(a) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the TEAD3 gene;
(b) an antisense nucleic acid against a transcript of the TEAD3 gene;
(c) a ribozyme nucleic acid against a transcription product of the TEAD3 gene;
(d) a dominant-negative mutant of TEAD3 or a nucleic acid encoding the same; or
(e) Decoy nucleic acid for TEAD3
That is, a drug . A method for producing iPS cells, comprising contacting a somatic cell with a nuclear reprogramming substance and an inhibitor of the transcription activation function of a group of genes that maintain the somatic cell identity of TEAD3,
The inhibitor is one of the following (a) to (e):
(a) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the TEAD3 gene;
(b) an antisense nucleic acid against a transcript of the TEAD3 gene;
(c) a ribozyme nucleic acid against a transcription product of the TEAD3 gene;
(d) a dominant-negative mutant of TEAD3 or a nucleic acid encoding the same; or
(e) Decoy nucleic acid for TEAD3
That's the method . The method according to claim 3 , wherein the nuclear reprogramming substances are Oct3/4, Klf4 and Sox2, or nucleic acids encoding the same. The method according to claim 3 , wherein the nuclear reprogramming substances are Oct3/4, Klf4, Sox2, and c-Myc, L-Myc, or N-Myc, or nucleic acids encoding them.