Multivalent Ligands to Control Stem Cell Fate

描述(申请人提供)：拟议工作的目标是设计有效的多价配体，以影响成人神经干细胞(NSCs)的分化和 基于多价仿生策略的人类多能干细胞(HPSCs)。细胞信号转导通常始于多价结合的配体，无论是分泌的还是细胞表面拴住的，与靶细胞受体结合，导致受体聚集。然而，目前控制多价相互作用从而调节生命系统内关键信号事件的能力非常有限。虽然已经实现了抗体诱导的配体或受体聚集，但这种方法并不能很好地控制、有效或容易重现。细胞内靶标可以通过重复诱导的二聚化结构域的小分子依赖的二聚化来聚集，但这种方法涉及过度表达融合蛋白，并且不容易适用于内源性配体或受体。使用合成的多价配体是一种很有前途的方法来控制和阐明细胞信号转导的基本机制。如果这种多价配体能够被设计成激活关键的信号通路，从而控制干细胞在体外和体内的命运，它们可以作为强大的生物工具和 作为一种有效的疗法。这项拟议工作的第一个目标是利用多价eaffin偶联物来研究eph-efrin信号调节神经干细胞和多能干细胞中细胞命运决定的机制。在这一目标下，我们将进行结构功能 分析在信号传递和干细胞分化中的多价性，以及在体外和体内设计有效激活Eph-EPhin信号的多价配体。我们的第二个目标是确定是否可以设计多价偶联物来激活NSCs中的Wnt信号，这将通过结合基于工程多肽的多价配体并在体外和体内表征它们的信号特性来实现。我们预计，由此产生的多价ePhin和Wnt配体将成为控制干细胞命运决定的有效生物活性材料，这一能力将对干细胞和发育生物学的机制研究具有重要意义，无论是在体外还是在体内。 至于应用方面，包括增强的细胞培养系统、药理学和毒理学筛选，以及恢复器官功能的再生医学方法。

DESCRIPTION (provided by applicant): The objective of the proposed work is to design potent multivalent ligands that influence the differentiation of adult neural stem cells (NSCs) and human pluripotent stem cells (hPSCs) based on a biomimetic strategy - multivalency. Cellular signal transduction can often begin with the multivalent binding of ligands, either secreted or cell-surface tethered, to target cell receptors, leading to receptor clustering. The capacity to control multivalent interactions and thereby modulate key signaling events within living systems is, however, currently very limited. While antibody-induced ligand or receptor clustering has been achieved, this method is not well- controlled, efficient, or readily reproducible. Intracellulr targets can be clustered by the small-molecule dependent dimerization of repeated inducible dimerizing domains, but this approach involves overexpressing fusion proteins and is not readily applicable for endogenous ligands or receptors. The use of synthetic multivalent ligands is a promising approach to control and to elucidate fundamental mechanisms in cellular signaling. If such multivalent ligands could be designed to activate key signaling pathways and thereby control stem cell fate in vitro and in vivo, they could serve as both powerful biological tools and as potent therapeutics. The first aim of the proposed work is to harness multivalent ephrin conjugates to study mechanisms by which Eph-ephrin signaling regulates cell fate decisions in neural stem cells and pluripotent stem cells. Within this aim, we will conduct a structure-function analysis of ephrin multivalency in signaling and stem cell differentiation, as well as engineer peptide-based multivalent ligands for potent activation of Eph-ephrin signaling in vitro and in vivo. Our second aim is to determine whether multivalent conjugates can be engineered to activate Wnt signaling in NSCs, which will be achieved with a combination of engineering peptide-based multivalent ligands and characterizing their signaling properties in vitro and in vivo. We anticipate that the resulting multivalent ephrin and Wnt ligands will serve as potent bioactive materials for controlling stem cell fate decisions, a capability that would be significan for mechanistic investigations in stem cell and developmental biology in vitro and in vivo, as well as for applications including enhanced cell culture systems, pharmacology and toxicology screens, and regenerative medicine approaches to restore organ function.