Quantitative Notch Signaling in Hematopoiesis

项目摘要 我们的实验室已经开发了使用工程化Notch配体的新型培养方法， 增加CD 34+前体的数量，包括那些能够提供快速造血的前体。 在接受脐带血（CB）移植的患者中植入。临床试验 确定了这种方法的安全性和有效性，但指出需要获得更大的 造血干细胞和祖细胞（HSPC）数量，以获得更有效、更经济的 可行的疗法因此，这项赠款的重点是阐明新的战略，以规范 Notch诱导的信号使HSPC生长相对于分化最大化。在这一点上，我们的CB CD 34+扩增方案集中于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Notch参数变化的贡献 表达和对激活的敏感性。最近的观察迫使我们调查 使用旁系同源物特异性活化抗体严格调节Notch信号强度水平 在细胞表面Notch表达的进化过程中产生， 文化（目标1）。这些研究还表明，针对这种病毒的旁系同源特异性抗体， Notch胞外区氨基端诱导干细胞中Notch活化 群体不易受Notch配体诱导的激活，通过克服抑制 内源性Notch配体表达导致的Notch激活（顺式抑制）。我们将确认 在促进Notch激活方面的差异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 增强可植入HSPC的产生（目的2）。这些研究将提供深入了解 Notch受体功能的细胞自主调节机制，概念性的结果， 和实际利益，那些专注于操纵细胞命运的决定，在Notch依赖 干细胞类型最后，我们将联合收割机结合我们的研究结果，最大限度地提高Notch诱导的HSPC自身， 更新，同时阻止分化，激活信号通路， HSPC存活，允许HSPC的进一步扩增（目的3）。这些目标共同推动我们 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扩增的实质性增加。

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.