Connexins in Mouse and Human Stem Cell Pluripotency

长期愿景：确定细胞间通讯在早期胚胎和整个发育过程中的作用；在多能干细胞的建立和维持中，在细胞重编程、谱系承诺和终末细胞命运规范中。***概述：多能干细胞具有向体内任何细胞类型分化的能力。细胞通讯对于协调干细胞存活、多能性和细胞命运规范所必需的复杂事件至关重要。间隙连接细胞间通讯（GJIC）通过密切相关的连接蛋白半通道实现相邻细胞之间的直接信号传递。GJIC在多能干细胞中的作用是有争议的：连接蛋白在小鼠干细胞中似乎是可有可无的，但在人类多能干细胞中却是必不可少的。我们现在知道，人类和小鼠干细胞在体外以不同的多能性状态存在。小鼠干细胞天生存在于多能性“中”基态，而人类多能性干细胞存在于更发育的高级状态，这是分化的“准备”状态。我们发现，药理学上的GJIC抑制杀死了启动的人类干细胞，但没有杀死。几种连接蛋白异构体（Cx30.3, Cx40, Cx43）在天然和引物多能干细胞中表达差异，表明异构体之间存在协同性。***短期目标：我们假设GJIC在原始多能干细胞中是可有可无的，但在细胞分化过程中则是必不可少的。我们将研究连接蛋白的表达和功能(1)在小鼠和人多能干细胞和(2)在这些多能干细胞的早期和晚期分化过程中。***目的1：研究干细胞多能性不同状态下连接蛋白的表达和功能。我们将使用CRISPR-Cas9基因消融技术检测连接蛋白敲除干细胞的干细胞增殖、凋亡和多能性。我们预计原始干细胞能够耐受连接蛋白基因的完全消融，而启动细胞则会死亡和/或失去其多能性潜能。***目的2：研究连接蛋白在体外分化早期和晚期的表达和功能。对照和连接素消融的干细胞将分化为三个胚胎胚层以及多能干细胞。分化为特化细胞类型将评估GJIC如何协调导致终端细胞命运规范的事件。我们预计cx消融的干细胞将表现出改变的分化潜能。***意义：我的长期愿景是了解控制细胞间通讯的基本分子机制，这对于释放小鼠和人类多能干细胞的潜力至关重要。这些研究代表了干细胞生物学的重要空白，揭示GJIC在细胞命运规范中的基本机制将拓宽我们对间隙连接的基本作用的认识。

Long-term vision: Defining the role of cell-cell communication in the early embryo and throughout development; in the establishment and maintenance of pluripotent stem cells, during cellular reprogramming, lineage commitment and terminal cell fate specification.***Overview: Pluripotent stem cells possess the ability to differentiate into any cell type in the body. Cellular communication is essential for coordinating the complex events necessary for stem cell survival, pluripotency and cell fate specification. Gap junctional intercellular communication (GJIC) enables direct signaling between neighboring cells through closely associated connexin hemichannels. The role of GJIC in pluripotent stem cells is controversial: connexins appear to be dispensable in mouse stem cells, but are conversely essential in human pluripotent stem cells. We now know that human and mouse stem cells inherently exist in different pluripotency states in vitro. Mouse stem cells innately exist in the pluripotent “nave” ground state while human pluripotent stem cells exist in a more developmentally advanced state which is “primed” for differentiation. We have found that pharmacological GJIC inhibition kills primed human stem cells but not nave. Several connexin isoforms (Cx30.3, Cx40, Cx43) are differentially expressed in nave and primed pluripotent stem cells, suggesting cooperativity between isoforms. ***Short term objectives: We hypothesize that GJIC is dispensable in nave pluripotent stem cells but becomes essential as cells are primed for differentiation. We will examine the expression and function of connexins (1) in nave and primed mouse and human pluripotent stem cells and (2) during early and late differentiation events of these pluripotent stem cells.***Aim1: Investigate connexin expression and function in differing states of stem cell pluripotency. We will examine stem cell proliferation, apoptosis and pluripotency in connexin knockout stem cells using CRISPR-Cas9 gene ablation. We expect that nave stem cells will tolerate complete connexin gene ablation while primed cells will die and/or lose their pluripotency potential. ***Aim2: Investigate connexin expression and function during early and late in vitro differentiation. Control and connexin-ablated stem cells will be differentiated toward the three embryonic germ layers as well as multipotent stem cells. Differentiation into specialized cell types will evaluate how GJIC coordinates events leading to terminal cell fate specification. We expect that Cx-ablated stem cells will exhibit altered differentiation potential. ***Significance: My long term vision of understanding the basic molecular mechanisms governing cell-cell communication is essential to unlocking the potential of mouse and human pluripotent stem cells. These studies represent important gaps in stem cell biology and uncovering basic mechanisms of GJIC during cell fate specification will broaden our appreciation of the fundamental role of gap junctions.