Connexins in Mouse and Human Stem Cell Pluripotency

长期视野：确定细胞-细胞通讯在早期胚胎和整个发育过程中的作用；在建立和维持多能干细胞过程中，在细胞重编程、谱系承诺和终末细胞命运指定过程中。*概述：多能干细胞具有分化为体内任何细胞类型的能力。细胞通讯对于协调干细胞存活、多能性和细胞命运指定所需的复杂事件至关重要。缝隙连接细胞间通讯(GJIC)通过紧密联系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存在争议：在小鼠干细胞中，连接蛋白似乎是必不可少的，但在人类多能干细胞中，连接蛋白似乎是必不可少的。我们现在知道，人和小鼠的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的“幼稚”基本状态，而人类多能干细胞存在于发育更高级的状态，为分化做好了“准备”。我们发现，药理上的GJIC抑制可以杀死启动的人类干细胞，但不能杀死NAVE。几种连接蛋白亚型(Cx30.3、Cx40、Cx43)在NAVE和启动的多能干细胞中有不同的表达，提示亚型之间存在协同作用。*短期目标：我们假设GJIC在NAVE多能干细胞中是必不可少的，但随着细胞为分化做好准备，GJIC变得必不可少。我们将研究缝隙连接蛋白的表达和功能：(1)在幼稚和启动的小鼠和人类多能干细胞中，以及(2)在这些多能干细胞的早期和晚期分化过程中。*目的：研究连接蛋白在干细胞多能性的不同状态下的表达和功能。我们将使用CRISPR-Cas9基因消融来检测连接蛋白基因敲除干细胞中的干细胞增殖、凋亡和多能性。我们预计，NAVE干细胞将耐受连接蛋白基因的完全去除，而启动的细胞将死亡和/或失去其多潜能。*AIM2：研究缝隙连接蛋白在体外分化早期和晚期的表达和功能。对照干细胞和去除连接蛋白的干细胞将分化为三个胚胎胚层和多能干细胞。分化成专门的细胞类型将评估GJIC如何协调导致终末细胞命运规范的事件。我们预计CX消融后的干细胞将显示出不同的分化潜能。*意义：我的长期愿景是了解支配细胞间交流的基本分子机制，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中的重要空白，揭示GJIC在细胞命运指定过程中的基本机制将扩大我们对缝隙连接基础作用的理解。

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.