Generation of induced pluripotent stem cells using chemical inhibition and three transcription factors

Methods Mol Biol. 2014;1150:227-36. doi: 10.1007/978-1-4939-0512-6_15.

Abstract

Generation of induced pluripotent stem (iPS) cells from differentiated cells has traditionally been performed by overexpressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. However, inclusion of c-Myc in the reprogramming cocktail can lead to expansion of transformed cells that are not fully reprogrammed, and studies have demonstrated that c-Myc reactivation increases tumorigenicity in chimeras and progeny mice. Moreover, chemical inhibition of Wnt signaling has been shown to enhance reprogramming efficiency. Here, we describe a modified protocol for generating iPS cells from murine fibroblasts using chemical inhibition and overexpression of three transcription factors. Using this protocol, we observed robust conversion to iPS cells while maintaining minimal contamination of partially reprogrammed transformed colonies.

MeSH terms

  • Animals
  • Cellular Reprogramming / drug effects*
  • Culture Media
  • Enzyme Inhibitors / pharmacology*
  • Glycogen Synthase Kinase 3 / antagonists & inhibitors
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / drug effects
  • Induced Pluripotent Stem Cells / metabolism
  • Kruppel-Like Factor 4
  • Mice
  • Retroviridae / genetics
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Culture Media
  • Enzyme Inhibitors
  • KLF4 protein, human
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Transcription Factors
  • Glycogen Synthase Kinase 3