title: Manipulation of KLF4 expression generates iPSCs paused at successive stages of reprogramming.
abstract: The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system) is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: ken-nishimura@md.tsukuba.ac.jp.
authors.forename: Ken
authors.initials: K
authors.lastname: Nishimura
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kato
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Lalhaba
authors.initials: L
authors.lastname: Oinam
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Shiomitsu
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ayakawa
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Manami
authors.initials: M
authors.lastname: Ohtaka
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Fukuda
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Mahito
authors.initials: M
authors.lastname: Nakanishi
authors.affiliation: Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: kojihisa@md.tsukuba.ac.jp.
authors.forename: Koji
authors.initials: K
authors.lastname: Hisatake
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D016000:Cluster Analysis; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D041681:NIH 3T3 Cells; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D049452:Green Fluorescent Proteins