Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.
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.
Papapetrou
Eirini P
EP
Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Tomishima
Mark J
MJ
Chambers
Stuart M
SM
Mica
Yvonne
Y
Reed
Evan
E
Menon
Jayanthi
J
Tabar
Viviane
V
Mo
Qianxing
Q
Studer
Lorenz
L
Sadelain
Michel
M
D002454:Cell Differentiation; D044127:Epigenesis, Genetic; D016259:Genes, myc / Q000502:physiology*; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000502:physiology*
C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors