人细胞重编程仍然极其低效,并且不同重编程因子的潜在机制是难以捉摸的。我们发现NANOG和LIN28(NL)协同作用可将OCT4,SOX2,KLF4和MYC(OSKM)介导的重编程提高约76倍,并将重编程潜伏期缩短至少1周。这种协同作用被GLIS1抑制,但被组蛋白甲基转移酶DOT1L(iDOT1L)的抑制剂增强,使TRA-1-60阳性()iPSC菌落增加〜127倍。机械上,NL是细胞上皮化重编程的主要驱动因素,Let-7miRNA靶LIN41的表达,以及经典WNT/β-CATENIN信号的激活,可以通过iDOT1L治疗进一步增强。除OSKM外,LIN41过表达同样促进细胞上皮化和WNT激活,显性阴性LIN41突变显著阻断NL和iDOT1L增强的重编程。我们还发现NL和iDOT1L诱导的典型WNT激活促进iPSC的初始发育动力学。然而,更成熟的大幅增加,通过在中后期重编程阶段抑制WNT活性,可以实现均匀的TRA-1-60集落形成。我们进一步发现,LIN41可以代替LIN28与NANOG协同作用,LIN41与NL的共表达进一步促进了WNT抑制下成熟iPSCs的形成。我们的研究确立了LIN41和典型的WNT信号作为NL的关键下游效应子,用于重编程效率和动力学的显着改善。并优化了从原代细胞稳健形成成熟人iPSC集落的条件。本文与该论文的第一作者进行了相关的第一人称访谈。
Human 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.