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.

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 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

  • GKLF protein
  • Homeodomain Proteins
  • 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