Abstract
ABSTRACTHuman cell reprogramming remains extremely inefficient and the underlying mechanisms by different reprogramming factors are elusive. We found that NANOG and LIN28 (NL) synergize to improve OCT4, SOX2, KLF4 and MYC (OSKM)-mediated reprogramming by ∼76-fold and shorten reprogramming latency by at least 1 week. This synergy is inhibited by GLIS1 but reinforced by an inhibitor of the histone methyltransferase DOT1L (iDOT1L) to a ∼127-fold increase in TRA-1-60-positive (+) iPSC colonies. Mechanistically, NL serve as the main drivers of reprogramming in cell epithelialization, the expression of Let-7 miRNA target LIN41, and the activation of canonical WNT/β-CATENIN signaling, which can be further enhanced by iDOT1L treatment. LIN41 overexpression in addition to OSKM similarly promoted cell epithelialization and WNT activation in reprogramming, and a dominant-negative LIN41 mutation significantly blocked NL- and iDOT1L-enhanced reprogramming. We also found that NL- and iDOT1L-induced canonical WNT activation facilitates the initial development kinetics of iPSCs. However, a substantial increase in more mature, homogeneous TRA-1-60+ colony formation was achieved by inhibiting WNT activity at the middle-to-late-reprogramming stage. We further found that LIN41 can replace LIN28 to synergize with NANOG, and that the coexpression of LIN41 with NL further enhanced the formation of mature iPSCs under WNT inhibition. Our study established LIN41 and canonical WNT signaling as the key downstream effectors of NL for the dramatic improvement in reprogramming efficiency and kinetics, and optimized a condition for the robust formation of mature human iPSC colonies from primary cells.This article has an associated First Person interview with the first author of the paper.
Highlights
Two gene cocktails, OCT4, SOX2, KLF4, and MYC (OSKM) (Takahashi et al, 2007; Takahashi and Yamanaka, 2006) and OCT4, SOX2, NANOG and LIN28A (OSNL) (Yu et al, 2007b), can reprogram somatic cells to embryonic stem cell (ESC)-like induced pluripotent stem cells
We found that NANOG and LIN28 (NL), but not GLIS1, synergize to stimulate the expression of the Let-7 target LIN41 and to enhance canonical WNT activity for human induced pluripotent stem cells (iPSCs) generation
NL is more efficient than GNL in reprogramming We first hypothesized that coexpressing GLIS1, NANOG and LIN28 would greatly enhance OSKM-mediated reprogramming based on their reported individual effects (Hanna et al, 2009; Lee et al, 2017; Maekawa et al, 2011; Yu et al, 2007b; Zhang et al, 2016)
Summary
OCT4, SOX2, KLF4, and MYC (OSKM) (Takahashi et al, 2007; Takahashi and Yamanaka, 2006) and OCT4, SOX2, NANOG and LIN28A (OSNL) (Yu et al, 2007b), can reprogram somatic cells to embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs). The early-to-middle reprogramming stages are characterized by multiple waves of lineagerelated gene activation in the order of developmental reversal, with MET occurring at the middle-to-late-reprogramming stage along with pluripotent network activation (Cacchiarelli et al, 2015). This transcriptional alteration in reprogramming is accompanied by epigenomic modifications that suppress somatic gene expression/ reactivation and maintain the active pluripotency regulatory network (Cacchiarelli et al, 2015; Hussein et al, 2014; Xu et al, 2016). The exact molecular mechanism that ensures successful human cell reprogramming is still poorly defined
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