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8-Bromo Cyclic Adenosine Monophosphate/*pharmacology; Animals; Cell Dedifferentiation/*drug effects; Cells, Cultured; Coculture Techniques; Fibroblasts/cytology/*drug effects; Gene Expression Profiling; Gene Expression Regulation/drug effects; Homeodomain Proteins/metabolism; Humans; Induced Pluripotent Stem Cells/cytology/*drug effects; Karyotyping; Male; Mice; Mice, SCID; Tumor Suppressor Protein p53/metabolism; Valproic Acid/pharmacology
Abstract:
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However, the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokine-related and inflammatory pathways. Remarkably, the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However, it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related, inflammatory pathways and self-renewal supporting gene, namely cyclin-encoding CCND2, and the associated genes CCNA1 and CCNE1. Conversely, we also see the down-regulation of the p53 (CCNB2, GTSE1, SERPINE1) and cell cycle (PLK1, CCNB2) pathways. Our data demonstrates that a cyclic AMP analog, 8-Br-cAMP, enhances the efficiency of cellular reprogramming. In addition, 8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming, which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming.
