Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

Eirini P. Papapetrou, Mark J. Tomishima, Stuart M. Chambers, Yvonne Mica, Evan Reed, Jayanthi Menon, Viviane Tabar, Qianxing Mo, Lorenz Studer, Michel Sadelain

Research output: Contribution to journalArticlepeer-review

237 Scopus citations

Abstract

Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, andwedemonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.

Original languageEnglish
Pages (from-to)12759-12764
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number31
DOIs
StatePublished - 4 Aug 2009
Externally publishedYes

Keywords

  • Fluorescent proteins
  • Lentiviral vectors
  • Silencing
  • Stoichiometry

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