The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering 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-beta 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.
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"This is consistent with our observation that ERK inhibition in TR-converted cells finalizes and/or stabilizes the transition of these cells to ESC pluripotency (Figures 2 and 3). Several kinase inhibitors have been reported either to support the generation of induced pluripotent stem (iPS) cells or to promote the conversion to ESC-like pluripotency (Ying et al., 2008; Li and Rana, 2012; Sato et al., 2004; Ichida et al., 2009; Maherali and Hochedlinger, 2009). Notably, TR did not inhibit GSK3beta, MEK/ERK, or ALK4/5, which are all targets of inhibitors that promote the reversion of EpiSCs into ESC-like cells (Greber et al., 2010; Zhou et al., 2010 ). "
[Show abstract][Hide abstract]ABSTRACT: It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.
"SB-431542 is a non-specific inhibitor of TGFβ receptor 1 kinase, and can also replace Sox2 and Oct4 protein during reprogramming . Furthermore, inhibition of TGFβ signaling early in reprogramming also alleviates the need for transgenic c-Myc expression [20,59]. The efficiency of generating iPSCs from human myoblasts is significantly improved by inhibitors of histone deacetylase (sodium butyrate) and TGFβ signaling (SB431542) . "
[Show abstract][Hide abstract]ABSTRACT: The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.
Full-text · Article · Feb 2016 · International Journal of Molecular Sciences
"The sustained expression of somatic cell pathways and suppression of mesenchymal-to-epithelial transition (MET) are two major barriers in the initial stages of reprogramming (Li et al., 2010; Samavarchi-Tehrani et al., 2010). Suppression of the transforming growth factorb (TGF-b) pathway or activation of the bone morphogenetic protein (BMP) pathway promote MET and increase reprogramming efficiency (Ichida et al., 2009; Samavarchi- Tehrani et al., 2010). The class of partially reprogrammed cells that successfully pass through the initial stages and fail to activate the endogenous pluripotency genes are referred to as pre-iPSCs (Theunissen et al., 2011). "
[Show abstract][Hide abstract]ABSTRACT: Reprogramming of somatic cells to generate induced pluripotent stem cells (iPSCs) has considerable latency and generates epigenetically distinct partially and fully reprogrammed clones. To understand the molecular basis of reprogramming and to distinguish the partially reprogrammed iPSC clones (pre-iPSCs), we analyzed several of these clones for their molecular signatures. Using a combination of markers that are expressed at different stages of reprogramming, we found that the partially reprogrammed stable clones have significant morphological and molecular heterogeneity in their response to transition to the fully pluripotent state. The pre-iPSCs had significant levels of OCT4 expression but exhibited variable levels of mesenchymal-to-epithelial transition. These novel molecular signatures that we identified would help in using these cells to understand the molecular mechanisms in the late of stages of reprogramming. Although morphologically similar mouse iPSC clones showed significant heterogeneity, the human iPSC clones isolated initially on the basis of morphology were highly homogeneous with respect to the levels of pluripotency.