Engineering a Carbohydrate-Biomaterial Interface for Control of Wnt Signaling and

描述（由申请人提供）：该项目的目标是操纵细胞表面的化学性质（通过新兴的基于代谢底物的低聚糖工程技术）和细胞结合的材料。通过使用这种方法，可以设计出高度兼容的粘附界面，支持基于碳水化合物的细胞粘附的新模式，并在粘附和信号通路之间建立密切的生化联系，为控制干细胞命运提供了一种创新的方法。具体来说，这一提议是基于“原理证明”的证明，即含硫醇的n -乙酰甘露糖胺（ManNAc）类似物“Ac5ManNTGc”可以阻断唾液酸的生物合成途径，转化为相应的含硫醇的“Neu5TGc”形式的唾液酸，然后在覆盖哺乳动物细胞表面的低聚糖中显示。一旦到达细胞表面，Neu5TGc通过反式二硫键促进宿主细胞与其他含硫醇细胞的附着，以及与化学相容的马来酰亚胺衍生或金表面的附着。重要的是，细胞在经历这种新的粘附模式后获得了新的行为；特别是Wnt信号通路在人类胚状体衍生（hEBD）干细胞中被激活，导致它们分化为神经元。第一个具体目标是专注于微调细胞表面的化学性质（通过改变巯基连接物到底层糖残基的长度，通过改变Neu5GTc表面密度，以及通过控制巯基氧化还原化学）和材料（通过图图化、微制造金和酰亚胺显示），以确定细胞-材料界面上有助于激活Wnt信号的精确因素。第二个具体目标将测试涉及ac5manntgc激活Wnt信号的遗传、分子和生化步骤的多步骤模型。前两个目标集中在一组表面化学（硫金/酰亚胺界面）和一条途径（Wnt信号）上，将提供详细的机制信息，为将“小分子寡糖工程”添加到组织工程工具包中提供坚实的科学基础。该方法有望广泛适用，为此，第三个具体目标(1)将评估含硫醇的甘油三酯类似物在第二胚胎干细胞系和成体干细胞中激活Wnt通路和神经分化的能力；(2)研究硫金/马来酰亚胺界面对涉及神经元分化的其他信号通路（g蛋白、TGF-¿和JNK）的影响。

DESCRIPTION (provided by applicant): The goal of this project is to manipulate the chemical properties of both the cell surface (through emerging metabolic substrate-based oligosaccharide engineering technology) and the material to which the cell binds. By using this approach, a highly-compatible adhesion interface can be engineered that supports novel modes of carbohydrate-based cell adhesion and - building on the intimate biochemical links between adhesion and signaling pathways - provides an innovative method to control stem cell fates. Specifically, this proposal is based on the "proof-of-principle" demonstration that the thiol-bearing N-acetylmannosamine (ManNAc) analog "Ac5ManNTGc" can intercept the sialic acid biosynthetic pathway, be converted into the corresponding thiol-bearing "Neu5TGc" form of sialic, and then be displayed in the oligosaccharides that cover the surfaces of mammalian cells. Once on the cell surface, Neu5TGc facilitates the attachment of the host cell to other thiol-bearing cells via trans-disulfide bonds as well as to chemically compatible maleimide- derivatized or gold surfaces. Importantly, cells gain new behaviors upon undergoing this novel mode of adhesion; in particular the Wnt signaling pathway was activated in human embryoid body-derived (hEBD) stem cells causing them to undergo differentiation to neurons. The first specific aim is focused on fine-tuning the chemical properties of the cell surface (by changing the thiol linker length to the underlying sugar residue, by changing Neu5GTc surface density, and by control of thiol redox chemistry) and material (by patterned, microfabricated gold and maleimide display) to determine the precise factors at the celhmaterial interface that contribute to activation of Wnt signaling. The second specific aim will test a multi-step model of the genetic, molecular and biochemical steps involved Ac5ManNTGc-activation of Wnt signaling. The first two aims, tightly focused on one set of surface chemistries (the thiohgold/maleimide interface) and one pathway (Wnt signaling), will provide detailed mechanistic information that will provide a solid scientific foundation for the addition of "small molecule oligosaccharide engineering" to the tissue engineering toolkit. This methodology is anticipated to be broadly applicable, and towards this end, the third specific aim (1) will evaluate the ability of thiol-bearing ManNAc analogs to activate the Wnt pathway and neural differentiation in a second embryonic stem cell line as well as in adult stem cells and (2) investigate the impact of thiohgold/maleimide interface on additional signaling pathways (G-protein, TGF-¿, and JNK) implicated in the differentiation of neurons.