1. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation
- Author
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Evan Reed, Eirini P. Papapetrou, Jayanthi Menon, Yvonne Mica, Lorenz Studer, Mark J. Tomishima, Michel Sadelain, Viviane Tabar, Stuart M. Chambers, and Qianxing Mo
- Subjects
Pluripotent Stem Cells ,Multidisciplinary ,SOXB1 Transcription Factors ,Cellular differentiation ,Genes, myc ,Kruppel-Like Transcription Factors ,Cell Differentiation ,Biological Sciences ,Biology ,Molecular biology ,Epigenesis, Genetic ,Cell biology ,Kruppel-Like Factor 4 ,Directed differentiation ,SOX2 ,KLF4 ,Humans ,Gene silencing ,Ectopic expression ,Induced pluripotent stem cell ,Octamer Transcription Factor-3 ,Reprogramming - 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, and we demonstrate 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.
- Published
- 2009