A continuous molecular roadmap to iPSC reprogramming through progression analysis of single-cell mass cytometry

Cell Stem Cell. 2015 Mar 5;16(3):323-37. doi: 10.1016/j.stem.2015.01.015.


To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4(high)Klf4(high) cells transitioned to a CD73(high)CD104(high)CD54(low) partially reprogrammed state. Ki67(low) cells from this intermediate population reverted to a MEF-like phenotype, but Ki67(high) cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like Nanog(high)Sox2(high)CD54(high) population and a mesendoderm-like Nanog(low)Sox2(low)Lin28(high)CD24(high)PDGFR-α(high) population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development.

Publication types

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

MeSH terms

  • Animals
  • Antigens, Differentiation* / biosynthesis
  • Antigens, Differentiation* / genetics
  • Cellular Reprogramming Techniques*
  • HEK293 Cells
  • Humans
  • Image Cytometry / methods*
  • Induced Pluripotent Stem Cells* / cytology
  • Induced Pluripotent Stem Cells* / metabolism
  • Kruppel-Like Factor 4
  • Mice
  • Transcription Factors* / biosynthesis
  • Transcription Factors* / genetics


  • Antigens, Differentiation
  • KLF4 protein, human
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Transcription Factors

Associated data

  • GEO/GSE56764