Principles of Signalling Pathway Modulation for Enhancing Human Naïve Pluripotency Induction [ChIP-seq]

Isolating human MEK/ERK signalling independent pluripotent stem cells (PSCs) with characteristics of naive pluripotency while maintaining differentiation competence and genetic integrity, remains challenging. Here we engineer reporter systems that allow screening for conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT-ßCATENIN, PKC and SRC signalling enables induction of teratoma competent naïve human PSCs, with capacity to differentiate into trophoblast stem cells (TSC) and extraembryonic endodermal (XEN) cells in vitro. Divergent signalling and transcriptional requirements for boosting naïve pluripotency were found between mouse and human. P53 depletion in human naïve PSCs endows them with competitiveness to better contribute to mouse-human cross-species chimeric embryos upon priming. Finally, MEK/ERK inhibition can be substituted with inhibition for NOTCH/RBPj, which allows inducing alternative human naïve-like PSCs with diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signalling foundations of human naïve pluripotency.

分离获得同时具备初始态多能性（naive pluripotency）特征、维持分化潜能与遗传完整性，且不依赖MEK/ERK信号通路的人类多能干细胞（Pluripotent Stem Cells, PSCs）仍极具挑战性。本研究构建了可用于筛选诱导人类初始态多能性分子与功能特征的培养条件的报告基因系统。通过协同抑制WNT/β-连环蛋白（WNT-βCATENIN）、蛋白激酶C（PKC）与Src（SRC）信号通路，可诱导出具备畸胎瘤形成能力的人类初始态多能干细胞（PSCs），该类细胞在体外可分化为滋养层干细胞（TSC）与胚外内胚层（XEN）细胞。研究发现，小鼠与人类细胞在增强初始态多能性所需的信号通路与转录调控需求方面存在显著差异。在人类初始态多能干细胞中敲除p53（P53），可赋予其竞争优势，使其在预激活后更高效地整合进入小鼠-人类跨物种嵌合胚胎。最后，可通过抑制NOTCH/RBPJ（NOTCH/RBPj）信号通路替代MEK/ERK信号通路的抑制，从而诱导出一类新型人类类初始态多能干细胞，该类细胞出现有害性全基因组DNA低甲基化的风险显著降低。本研究结果为阐明人类初始态多能性的信号调控基础提供了研究框架。