ABSTRACT
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is accompanied by morphological, functional, and metabolic alterations before acquisition of full pluripotency. Although the genome-wide effects of the reprogramming factors on gene expression are well documented, precise mechanisms by which gene expression changes evoke phenotypic responses remain to be determined. We used a Sendai virus-based system that permits reprogramming to progress in a strictly KLF4-dependent manner to screen for KLF4 target genes that are critical for the progression of reprogramming. The screening identified Tcl1 as a critical target gene that directs the metabolic shift from oxidative phosphorylation to glycolysis. KLF4-induced TCL1 employs a two-pronged mechanism, whereby TCL1 activates AKT to enhance glycolysis and counteracts PnPase to diminish oxidative phosphorylation. These regulatory mechanisms described here highlight a central role for a reprogramming factor in orchestrating the metabolic shift toward the acquisition of pluripotency during iPSC generation.
Subject(s)
Cellular Reprogramming , Energy Metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Transcription Factors/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cellular Reprogramming/genetics , Gene Expression Profiling , Gene Expression Regulation , Glycolysis , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mitochondria/genetics , Mitochondria/metabolism , Oxidative Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system) is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.
