A Small-Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog

Abstract

Summary The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem (iPS) cells. However, the transgenic methods used to deliver reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-β signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

Two-Phase Analysis of Molecular Pathways Underlying Induced Pluripotent Stem Cell Induction

“…The important role of Sox2 in the second phase of reprogramming is also consistent with the main finding of a recent study that has identified a small molecule (RepSox) able to replace Sox2 for reprogramming [24]. In agreement with our work, this study showed that inhibition of TGF-b signaling is critical to RepSox's replacement of Sox2 in iPSC induction.…”

“…This unexpected finding was consistent with our hypothesis that a stable intermediate phase between fibroblasts and iPSCs could be achieved through the expression of only three reprogramming factors (Oct4, Klf4, and c-Myc). In fact, similar colonies were present in the original discovery [24,32], although subsequently identified differences in potential for reprogramming set these apart [21][22][23].…”

“…One main difference between this study and our own is our inability to replace Sox2 with Nanog. Although Nanog was able to replace Sox2 in their work [24], cells in the colonies obtained by Nanog overexpression showed limited differentiation ability. Our results are in line with recent findings showing late induction of Nanog, even after overexpression of all four Yamanaka factors [38].…”

“…cells and iPSCs may not be critical for the reprogramming process, as, by definition, these cells fail to become fully reprogrammed iPSCs. Recent identifications of small molecules capable of catalyzing or even replacing certain reprogramming factors provide an alternative means of analyzing the role of these four reprogramming factors in iPSC induction [22,[24][25][26][27]. Complicating this method, however, is the fact that small chemical compounds can bind to multiple targets and act on multiple signaling pathways that may not be relevant to the reprogramming process.…”

Induction of pluripotency by defined factors

“…The treatment with Aza-C or the suppression of the Dnmt1 level induces maturation from partially reprogrammed cells to completely reprogrammed iPS cells. 68), 69) Mesenchymal-to-epithelial transition. During the iPS cell induction from fibroblasts, cells undergo morphological changes that resemble the mesenchymal-to-epithelial Transition (MET).…”