Novel regulation of Notch-induced HSPC expansion

项目摘要 我们的实验室已经开发了新的培养方法，使用工程Notch配体来增加 CD 34+前体，包括那些能够在接受造血干细胞移植的患者中提供快速造血移植的前体。 脐带血移植。临床试验表明，需要获得更大的造血干细胞， 祖细胞（HSPC）数量，以获得更有效、经济上可行的治疗。因此，这笔赠款 旨在阐明调控Notch诱导的信号强度以最大化HSPC生长的新策略， 利用1）HSPC亚群中Notch受体对活化的敏感性和2）Notch 促进HSPC扩增相对于T细胞分化所需的信号强度。为了实现这一目标，我们 已经确定，针对Notch胞外结构域的氨基末端产生的抗体比针对Notch胞外结构域的氨基末端产生的抗体更多。 在激活原始HSPC上的受体方面比Notch配体更有效，从而导致增加的In-Fos的产生。 体内细胞重建;初步研究表明这是抗体介导的细胞颠覆的结果 原始HSPC中内源性表达配体施加的抑制作用（顺式抑制）。在Aim 1，我们测试了我们的假设，即在促进Notch活化和HSPC生成方面的差异激动剂效力 结果来自配体介导的顺式抑制。我们将研究抑制性配体在CB中的表达和功能 使用shRNA敲低和/或阻断抗体处理连同移植测定的HSPC。到 阐明抗体颠覆顺式抑制的机制，指导未来改良的发展 在使用激动剂时，我们将确定靶表位位置和增加的激动剂亲和力的贡献。我们 先前的研究还确定了产生Notch信号的不同水平的要求， HSPC与促进T细胞分化。因此，在目标2中，我们测试了我们的假设，即使用paradigm- 特异性Notch抗体激动剂将微调Notch活化水平， 产生骨髓和胸腺再生细胞。我们将调查低密度的动态使用， Notch旁系同源物特异性抗体激动剂通过诱导进一步增加骨髓再生细胞生成 在顺式抑制的原始前体中，Notch激活，但维持低水平的Notch信号强度， 防止这些细胞转向T细胞系。我们进一步探讨了旁系同源特异性抗体激动剂 通过诱导不同水平的Notch， 在不同的造血亚群中激活。总的来说，这些研究将提供对细胞的深入了解- 自主机制调节Notch受体功能，概念和实际利益的结果 对于那些专注于在Notch依赖性干细胞类型中操纵细胞命运决定的人，这里最相关的， HSPC和前胸腺细胞前体的扩增用于开发可行的临床治疗剂。

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 indicate the need to derive greater hematopoietic stem and progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, this grant aims to elucidate new strategies to regulate Notch-induced signal strength to maximize HSPC growth, focusing on exploiting differences in 1) Notch receptor susceptibility to activation among HSPC subsets and 2) Notch signal strength necessary to promote HSPC expansion versus T cell differentiation. Towards this goal, we have determined that antibodies raised against the amino-terminus of the Notch extracellular domain are more effective than Notch ligands in activating receptors on primitive HSPC, leading to increased generation of in vivo repopulating cells; preliminary studies have suggested this results from antibody-mediated subversion of inhibitory effects imposed by endogenously expressed ligands in primitive HSPC (cis-inhibition). Thus, in Aim 1, we test our hypothesis that differential agonist potency in promoting Notch activation and HSPC generation results from ligand-mediated cis-inhibition. We will investigate inhibitory ligand expression and function in CB HSPC using shRNA knockdown and/or blocking antibody treatment together with transplantation assays. To elucidate the mechanism whereby antibody subverts cis-inhibition, guiding the future development of improved agonists, we will determine the contribution of target epitope location and increased agonist affinity. Our previous studies have also determined requirements for distinct levels of Notch signaling for generation of HSPC versus promotion of T cell differentiation. Thus, in Aim 2, we test our hypothesis that the use of paralog- specific Notch antibody agonists will fine-tune the level of Notch activation to concurrently enhance the generation of marrow and thymic repopulating cells. We will investigate the dynamic use of low densities of Notch paralog-specific antibody agonists to further increase marrow repopulating cell generation by inducing Notch activation in cis-inhibited primitive precursors, yet maintain a low level of Notch signal strength to prevent diversion of these cells to the T-lineage. We further explore whether paralog-specific antibody agonists can simultaneously generate marrow and thymic repopulating cells by inducing different levels of Notch activation in different hematopoietic subsets. Overall, 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, most relevant here, the expansion of HSPC and prothymocyte precursors for the development of viable clinical therapeutics.