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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 31; pp. 12759 - 12764
• Main Authors: Papapetrou, Eirini P, Tomishima, Mark J, Chambers, Stuart M, Mica, Yvonne, Reed, Evan, Menon, Jayanthi, Tabar, Viviane, Mo, Qianxing, Studer, Lorenz, Sadelain, Michel
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 04.08.2009; National Acad Sciences
• Subjects: Biological Sciences; c-Myc protein; Cell Differentiation; Cell lines; Differentiation; Epigenesis, Genetic; Expression vectors; Fibroblasts; Fluorescence; Gene expression; Genes, myc - physiology; Genetic vectors; Geometric lines; Humans; Induced pluripotent stem cells; Inhibitory postsynaptic potentials; KLF4 protein; Kruppel-Like Transcription Factors - genetics; Kruppel-Like Transcription Factors - physiology; Oct-4 protein; Octamer Transcription Factor-3 - genetics; Octamer Transcription Factor-3 - physiology; Pluripotent stem cells; Pluripotent Stem Cells - cytology; Proteins; Somatic cells; SOXB1 Transcription Factors - genetics; SOXB1 Transcription Factors - physiology; Stem cells; Stoichiometry; Transgenes
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0904825106

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.