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

描述（由申请人提供）：本项目的目标是操纵细胞表面（通过新兴的基于代谢底物的寡糖工程技术）和细胞结合材料的化学性质。通过使用这种方法，可以设计高度相容的粘附界面，其支持基于碳水化合物的细胞粘附的新模式，并且-建立在粘附和信号传导途径之间的密切生化联系上-提供了控制干细胞命运的创新方法。具体而言，该提议基于“原理证明”，即带有巯基的N-乙酰甘露糖胺（ManNAc）类似物“Ac 5 ManNTGc”可以阻断唾液酸生物合成途径，转化为相应的带有巯基的“Neu 5 TGc”形式的唾液酸，然后展示在覆盖哺乳动物细胞表面的寡糖中。一旦在细胞表面上，Neu 5 TGc促进宿主细胞通过反式二硫键附着到其他携带巯基的细胞以及化学相容的马来酰亚胺衍生或金表面。重要的是，细胞在经历这种新的粘附模式后获得新的行为;特别是Wnt信号通路在人胚状体衍生的（hEBD）干细胞中被激活，使它们分化为神经元。第一个具体目标集中在微调细胞表面的化学性质（通过改变硫醇连接基长度到下面的糖残基，通过改变Neu 5GTc表面密度，并通过控制硫醇氧化还原化学）和材料（通过图案化的，微加工的金和马来酰亚胺显示），以确定在细胞材料界面的精确因素，有助于激活Wnt信号传导。第二个具体目标将测试涉及Wnt信号传导的Ac 5 ManNTGc激活的遗传、分子和生物化学步骤的多步骤模型。前两个目标，紧紧集中在一组表面化学（thiohgold/马来酰亚胺界面）和一个途径（Wnt信号），将提供详细的机制信息，将提供一个坚实的科学基础，增加“小分子寡糖工程”的组织工程工具包。该方法预期是广泛适用的，并且为此，第三个具体目标（1）将评估带有巯基的ManNAc类似物在第二胚胎干细胞系以及在成体干细胞中激活Wnt途径和神经分化的能力，以及（2）研究硫代金/马来酰亚胺界面对另外的信号传导途径的影响（G蛋白，TGF-β和JNK）参与神经元的分化。

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.