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. Overall design: ChIP-seq of the following proteins: H3K27ac, KLF17, KLF4, NANOG, OCT4, SOX2 and TFAP2C, measured in human pluropotent cells (PSCs) maintained in naïve conditions (Enhanced-NHSM), or in conventional/primed human PSCs, Measured on various cell lines.

获取具备初始多能性（naive pluripotency）特征、同时维持分化潜能与遗传完整性的、不依赖MEK/ERK信号通路的人类多能干细胞（PSCs），仍是一项极具挑战性的研究课题。本研究构建了可用于筛选诱导人类初始多能性分子与功能特征培养条件的报告基因系统。协同抑制WNT/β-连环蛋白（WNT-βCATENIN）、蛋白激酶C（PKC）及SRC信号通路，可诱导出具备畸胎瘤形成能力的初始态人类多能干细胞，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，小鼠与人类在增强初始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，可提升其竞争能力，使其在经历多能性启动（priming）后，更高效地参与人-小鼠跨物种嵌合胚胎的构建。此外，可通过抑制NOTCH/RBPj通路替代MEK/ERK通路的抑制，从而诱导出另一类人类初始样多能干细胞，这类细胞发生全局性有害DNA低甲基化的风险显著降低。本研究结果为阐明人类初始多能性的信号调控基础提供了理论框架。整体实验设计：对以下蛋白质进行染色质免疫共沉淀测序（ChIP-seq）：H3K27乙酰化修饰（H3K27ac）、KLF17、KLF4、NANOG、OCT4、SOX2及TFAP2C；检测样本分别为维持在初始态培养条件（Enhanced-NHSM）下的人类多能干细胞，以及常规/始发态人类多能干细胞，实验覆盖多种细胞系。