Quantitative Notch Signaling in Hematopoiesis

项目总结 我们的实验室已经开发出使用工程Notch配体的新型培养方法 增加CD34+前体细胞的数量，包括那些能够提供快速造血的细胞 脐带血移植患者的植入。临床试验已经 确定了此方法的安全性和有效性，但表示需要派生出更大的 造血干细胞和祖细胞(HSPC)数量更强大，更经济 可行的治疗方法。因此，这笔拨款的重点是阐明监管的新战略 缺口诱导信号，以最大限度地促进HSPC生长与分化。到目前为止，我们的CB CD34+扩展协议的重点是通过控制配体来调节Notch信号强度 可用性，而在很大程度上忽略了细胞表面凹陷平行曲线中的变异的贡献 表达和对激活的敏感性。最近的观察迫使我们调查 利用类对数特异性激活抗体严密调控Notch信号强度水平 在伴随着体外细胞表面Notch表达的进化过程中产生 文化(目标1)。这些研究还表明，针对PARALLOG特异性抗体的 Notch胞外区的氨基末端诱导干细胞中的Notch激活 通过克服Notch配体的抑制而使不受Notch配体诱导的激活的群体 内源性Notch配体表达引起的Notch激活(顺式抑制)。我们会确认 促进Notch激活的不同激动剂效力源于配体介导的顺式- 抑制并确定是否使用Notch抗体激动剂来克服这种抑制 增强可刻HSPC的生成(目标2)。这些研究将提供对 调控Notch受体功能的细胞自主机制是概念性的结果 以及那些专注于在Notch-Dependent中操纵细胞命运决定的人的实际兴趣 干细胞类型。最后，我们将结合我们的研究结果，最大化Notch诱导的HSPC自我 在抑制分化的同时激活促进分化的信号通路 HSPC生存，允许HSPC进一步扩张(目标3)。这些目标共同推动了我们 我们的长期目标是开发干细胞工程的新策略，重点是 实现用于治疗目的的HSPC扩容的大幅增加。

PROJECT SUMMARY Our laboratory has developed novel culture methods using engineered Notch ligands to increase the number of CD34+ precursors, including those able to provide rapid hematopoietic engraftment in patients undergoing cord blood (CB) transplantation. Clinical trials have determined the safety and effectiveness of this approach, but indicate the need to derive greater hematopoietic stem and progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, the focus of this grant is to elucidate new strategies to regulate Notch-induced signals to maximize HSPC growth versus differentiation. To this point, our CB CD34+ expansion protocol has focused on regulating Notch signal strength by controlling ligand availability, while largely neglecting the contribution of variation in cell-surface Notch paralog expression and susceptibility to activation. Recent observations compel us to investigate the use of paralog-specific activating antibodies to tightly regulate the level of Notch signal strength generated during the evolution of cell surface Notch expression that accompanies ex vivo culture (Aim 1). These studies also suggest that paralog-specific antibodies raised against the amino-terminus of the Notch extracellular domain induce Notch activation in stem cell populations insusceptible to Notch ligand-induced activation by overcoming the inhibition of Notch activation due to endogenous Notch ligand expression (cis-inhibition). We will confirm that differential agonist potency in promoting Notch activation stems from ligand-mediated cis- inhibition and determine whether use of Notch antibody agonists to overcome this suppression enhances the generation of engraftable HSPC (Aim 2). These studies will provide insight into the cell-autonomous mechanisms regulating Notch receptor function, an outcome of conceptual and practical interest to those focused on manipulating cell-fate decisions in Notch-dependent stem cell types. Lastly, we will combine our findings maximizing Notch-induced HSPC self- renewal while preventing differentiation with the activation of signaling pathways promoting HSPC survival, allowing further expansion of HSPC (Aim 3). Together, these aims advance us toward our long-term goal of developing novel strategies for stem cell engineering, with a focus on achieving a substantial increase in HSPC expansion for therapeutic purposes.