Manipulation of KLF4 expression generates iPSCs paused at successive stages of reprogramming

Ken Nishimura; Tetsuo Kato; Chen Chen; Lalhaba Oinam; Emi Shiomitsu; Daisuke Ayakawa; Manami Ohtaka; Aya Fukuda; Mahito Nakanishi; Koji Hisatake

doi:10.1016/j.stemcr.2014.08.014

Manipulation of KLF4 expression generates iPSCs paused at successive stages of reprogramming

Stem Cell Reports. 2014 Nov 11;3(5):915-29. doi: 10.1016/j.stemcr.2014.08.014. Epub 2014 Oct 2.

Authors

Affiliations

¹ 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.
² Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
³ Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
⁴ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Blotting, Western
Cells, Cultured
Cellular Reprogramming / genetics*
Cluster Analysis
Embryo, Mammalian / cytology
Fibroblasts / cytology
Fibroblasts / metabolism
Gene Expression Profiling*
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Homeodomain Proteins / genetics
Homeodomain Proteins / metabolism
Humans
Induced Pluripotent Stem Cells / metabolism*
Kruppel-Like Factor 4
Kruppel-Like Transcription Factors / genetics*
Kruppel-Like Transcription Factors / metabolism
Mice
Mice, Inbred C57BL
Mice, Transgenic
NIH 3T3 Cells
Nanog Homeobox Protein
Octamer Transcription Factor-3 / genetics
Octamer Transcription Factor-3 / metabolism
Oligonucleotide Array Sequence Analysis
Proto-Oncogene Proteins c-myc / genetics
Proto-Oncogene Proteins c-myc / metabolism
Reverse Transcriptase Polymerase Chain Reaction
SOXB1 Transcription Factors / genetics
SOXB1 Transcription Factors / metabolism

Substances

Homeodomain Proteins
KLF4 protein, human
Klf4 protein, mouse
Kruppel-Like Factor 4
Kruppel-Like Transcription Factors
Nanog Homeobox Protein
Nanog protein, mouse
Octamer Transcription Factor-3
Pou5f1 protein, mouse
Proto-Oncogene Proteins c-myc
SOXB1 Transcription Factors
Sox2 protein, mouse
Green Fluorescent Proteins

Associated data

GEO/GSE56406