Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming

Nat Cell Biol. 2013 Jul;15(7):829-38. doi: 10.1038/ncb2765. Epub 2013 May 26.

Abstract

Present practices for reprogramming somatic cells to induced pluripotent stem cells involve simultaneous introduction of reprogramming factors. Here we report that a sequential introduction protocol (Oct4-Klf4 first, then c-Myc and finally Sox2) outperforms the simultaneous one. Surprisingly, the sequential protocol activates an early epithelial-to-mesenchymal transition (EMT) as indicated by the upregulation of Slug and N-cadherin followed by a delayed mesenchymal-to-epithelial transition (MET). An early EMT induced by 1.5-day TGF-β treatment enhances reprogramming with the simultaneous protocol, whereas 12-day treatment blocks reprogramming. Consistent results were obtained when the TGF-β antagonist Repsox was applied in the sequential protocol. These results reveal a time-sensitive role of individual factors for optimal reprogramming and a sequential EMT-MET mechanism at the start of reprogramming. Our studies provide a rationale for further optimizing reprogramming, and introduce the concept of a sequential EMT-MET mechanism for cell fate decision that should be investigated further in other systems, both in vitro and in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cadherins / metabolism
  • Cell Differentiation
  • Cells, Cultured
  • Cellular Reprogramming*
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism
  • Epithelial-Mesenchymal Transition*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Green Fluorescent Proteins / metabolism
  • Immunoblotting
  • Induced Pluripotent Stem Cells / metabolism
  • Induced Pluripotent Stem Cells / pathology*
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / metabolism*
  • Mice
  • Octamer Transcription Factor-3 / metabolism*
  • Proto-Oncogene Proteins c-myc / metabolism*
  • Pyrazoles / pharmacology
  • Pyridines / pharmacology
  • SOXB1 Transcription Factors / metabolism*
  • Transforming Growth Factor beta / metabolism
  • Wound Healing

Substances

  • Cadherins
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Proto-Oncogene Proteins c-myc
  • Pyrazoles
  • Pyridines
  • RepSox
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse
  • Transforming Growth Factor beta
  • Green Fluorescent Proteins