The role of intracellular pH in the specification of cell fate

胚胎和成人中的干细胞有能力转变为特定的细胞类型，例如制造胰岛素的胰腺细胞，吸收营养的肠细胞，以及可以成为免疫细胞或携带氧气的细胞的主血细胞。干细胞如何改变它们的身份对于理解人类发育、发育缺陷和再生医学方法非常重要。在发育方面，研究的目的是了解胚胎干细胞如何成为制造器官（如心脏和肝脏）的各种细胞类型。发育“出错”往往是导致唇腭裂和心脏畸形等缺陷以及癌症和糖尿病等疾病的原因。再生医学有望修复受损器官或治疗阿尔茨海默氏症等疾病。因此，干细胞如何改变它们的身份或“命运”是一个非常重要的问题。这个问题的一个新答案是，当干细胞改变其身份时，它们会改变其内部的酸碱平衡。目前的提案测试了关于酸碱平衡的这些变化如何确保正确细胞命运的预测。预测将通过使用新的工具来测试，以准确地测量活的分离的干细胞和整个动物的干细胞中的酸碱平衡，并通过使用新的计算和实验方法来了解酸碱平衡的变化如何改变蛋白质的形状和功能，以及以前公认的控制细胞命运的基因的表达。关于细胞命运如何从幼稚干细胞中指定的研究主要集中在信号通路，转录程序和表观遗传变化的调节。新的研究结果表明，细胞内pH（pHi）动态是一个以前未被认识到的关键调节剂的干细胞的命运在三个干细胞模型：小鼠胚胎干细胞（mESC），成年果蝇卵泡干细胞（FSC），和成年小鼠肠干细胞（ISC）。建立在这些发现将产生一个机制的理解如何pHi动力学指定细胞的命运。目的1解决了如何pHi动力学调节mESC多能性和FSC分化的假设，即pHi调节不同的干细胞状态，通过pHi依赖性的活性或配体结合亲和力的选择性内源性蛋白质的作用，在调节细胞命运的决定。已知的（β-连环蛋白，DIDO 3，磷酸果糖激酶-1）和预测的（BCL 9和FOXM 1）pH敏感蛋白的作用将在分子，细胞和动物尺度上解决。目的2通过使用ISC类器官的研究确定细胞命运决定中pHi动力学的保守性。初步数据支持测试的假设，即ISCs的pH依赖性分化发生在两个步骤：隐窝出芽和谱系规范。将通过关注pH调节的放线菌球蛋白收缩性和Wnt信号传导来确定隐窝出芽，并且将通过使用谱系特异性荧光报告物和Wnt和Notch途径活性的报告物来解析分泌细胞的谱系特化。结果将产生关于pHi动力学如何调节细胞命运决定的机制性见解，这对了解人类发育，发育缺陷和再生医学方法具有重要意义。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

Non-Technical Paragraph Stem cells in embryos and adults have the capacity to change to specific cell types, such as pancreatic cells that make insulin, intestinal cells that absorb nutrients, and master blood cells that can become an immune cell or a cell that carries oxygen. How stem cells change their identity is important for understanding human development, developmental defects, and approaches for regenerative medicine. For development, research is aimed at understanding how an embryonic stem cell becomes all the diverse cell types that make organs, like the heart and liver. Development “gone wrong” is often the cause of defects such as cleft-lip palate and heart malformations, as well as diseases such as cancer and diabetes. And regenerative medicine holds promise to repair damaged organs or treat diseases like Alzheimer’s. Hence, how stem cells change their identity or “fate” is a highly significant question to resolve. One new answer to this question is that when stem cells change their identity they change their internal acid-base balance. The current proposal tests predictions on how these changes in acid-base balance ensure a correct cell fate. Predictions will be tested by using new tools to accurately measure acid-base balance in live isolated stem cells and in stem cells in whole animals, and by using new computational and experimental approaches to understand how changes in acid-base balance change the shape and function of proteins as well as the expression of genes previously recognized for controlling how a cell fate is specified. Technical Paragraph Studies on how cell fate is specified from naïve stem cells mostly focus on regulation by signaling circuits, transcriptional programs, and epigenetic changes. New findings reveal that intracellular pH (pHi) dynamics is a previously unrecognized critical regulator of stem cell fates in three stem cell models: mouse embryonic stem cells (mESCs), adult Drosophila follicle stem cells (FSCs), and adult mouse intestinal stem cells (ISCs). Building on these findings will generate a mechanistic understanding of how pHi dynamics specifies cell fate. Aim 1 addresses how pHi dynamics regulates mESC pluripotency and FSC differentiation by testing the hypothesis that pHi regulates distinct stem cell states through pHi-dependent effects on the activity or ligand-binding affinity of selective endogenous proteins with established roles in regulating cell fate decisions. Roles for known (-catenin, DIDO3, phosphofructokinase-1) and predicted (BCL9 and FOXM1) pH sensitive proteins will be resolved at molecular, cellular and animal scales. Aim 2 determines the conservation of pHi dynamics in cell fate decisions through studies using ISC organoids. Preliminary data support testing the hypothesis that pHi-dependent differentiation of ISCs occurs at two steps; crypt budding and lineage specification. Crypt budding will be determined by focusing on pHi-regulated actinomyosin contractility and Wnt signaling, and lineage specification to secretory cells will be resolved by using lineage-specific fluorescent reporters and reporters for Wnt and Notch pathway activity. Outcomes will generate mechanistic insights on how pHi dynamics regulates cell fate decisions that will be significant to understand human development, developmental defects, and approaches for regenerative medicine.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.