Two-phase analysis of molecular pathways underlying induced pluripotent stem cell induction

Stem Cells. 2011 Dec;29(12):1963-74. doi: 10.1002/stem.752.

Abstract

Induced pluripotent stem cells (iPSCs) can be reprogrammed from adult somatic cells by transduction with Oct4, Sox2, Klf4, and c-Myc, but the molecular cascades initiated by these factors remain poorly understood. Impeding their elucidation is the stochastic nature of the iPS induction process, which results in heterogeneous cell populations. Here we have synchronized the reprogramming process by a two-phase induction: an initial stable intermediate phase following transduction with Oct4, Klf4, and c-Myc, and a final iPS phase following overexpression of Sox2. This approach has enabled us to examine temporal gene expression profiles, permitting the identification of Sox2 downstream genes critical for induction. Furthermore, we have validated the feasibility of our new approach by using it to confirm that downregulation of transforming growth factor β signaling by Sox2 proves essential to the reprogramming process. Thus, we present a novel means for dissecting the details underlying the induction of iPSCs, an approach with significant utility in this arena and the potential for wide-ranging implications in the study of other reprogramming mechanisms.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • Cells, Cultured / cytology
  • Female
  • Fibroblasts / cytology
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Immunohistochemistry
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Nude
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / metabolism
  • Plasmids / genetics
  • Plasmids / metabolism
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • Retroviridae / genetics
  • Retroviridae / metabolism
  • SOXB1 Transcription Factors / genetics
  • SOXB1 Transcription Factors / metabolism*
  • Signal Transduction*
  • Teratoma / genetics
  • Teratoma / metabolism
  • Teratoma / pathology
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism*

Substances

  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Myc protein, mouse
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Proto-Oncogene Proteins c-myc
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse
  • Transforming Growth Factor beta