Nanoscale Assembly of Bioactive Ligands to Enhance Endothelial Differentiation

项目摘要 内皮细胞具有重要的生物医学应用，其范围从增强工程化血管的开放性， 血管移植物和支架以促进缺血组织中的新血管形成。但它们的有限供应 阻碍了内皮细胞相关技术的成功。干细胞技术的进步提供了一个 解决这个问题的独特机会。特别地，内皮细胞已经来源于人类， 多能干细胞（hPSC），其可以广泛增殖并且实际上提供无限的细胞来源。 最近在制备诱导的PSC（iPSC）方面的成功提供了额外的优势， 免疫相容的自体hPSC，并在未来实现“个性化”治疗。的关键 利用这个机会来推进内皮细胞相关技术是我们引导内皮细胞的能力， 分化在目前使用的方法中，将hPSC分化成血管细胞，其具有两种细胞特性。 造血和内皮细胞的潜力，然后成血管细胞分化为内皮细胞， VEGF和纤连蛋白（FN）包被表面的存在。VEGF和FN都是有效的 内皮细胞分化，并且由于FN的独特结构，它们表现出协同作用， 细胞粘附位点和VEGF结合位点以纳米级接近定位。然而，天然衍生的FN 不同批次之间有差异。此外，共价固定的FN具有阻断细胞的结构变化， 粘附配体;物理吸附的FN保留了活性细胞粘附结构域，但不允许精确的 控制表面配体密度。因此，用FN产生的细胞微环境没有受到严格控制， 阻碍了从干细胞持续产生内皮细胞。这个问题可以通过使用 良好控制的合成材料，概括了协同作用的基本分子结构， VEGF和FN在调节内皮细胞分化中的作用。该应用程序的目标是开发 具有VEGF协同作用的基本结构特征的合成材料， FN，并使用这些材料来引导人iPSC衍生的成血管细胞的内皮分化。我们 中心假设是细胞粘附肽和VEGF模拟肽融合到一对 异二聚卷曲螺旋分别可以通过卷曲螺旋自聚合而进入纳米级接近。 组装和用异二聚体官能化的材料以及可溶性因子将产生良好的- 用于iPSC衍生的内皮细胞的有效和可再现的内皮分化的受控细胞微环境 成血管细胞本论文的具体目标是：（1）设计、合成、表征和鉴定多肽 其自组装以纳米级接近呈现细胞粘附肽和VEGF模拟肽;（2） 在多肽功能化的细胞上检查人iPSC衍生的成血管细胞的内皮分化。 印刷受体.该项目的成功完成将导致良好的控制，仿生细胞 在微环境中，诱导iPSC衍生的成血管细胞的有效和稳健的内皮分化。

Project Abstract Endothelial cells have important biomedical applications ranging from enhancing the patency of engineered vascular grafts and stents to promoting neovascularization in ischemic tissues. But their limited availability hinders the success of endothelial-cell-related technologies. The advances in stem cell technology offer a unique opportunity to address this issue. In particular, endothelial cells have been derived from human pluripotent stem cells (hPSCs), which can proliferate extensively and virtually provide an unlimited cell source. The recent success in making induced PSCs (iPSCs) offers additional advantages in providing immunologically compatible autologous hPSCs and enabling "personalized" therapy in the future. The key to exploiting this opportunity to advance endothelial-cell-related technologies is our ability to guide endothelial differentiation. In currently used methods, hPSCs are differentiated into hemangioblasts, which have both hematopoietic and endothelial potentials, followed by differentiation of hemangioblasts into endothelial cells in the presence of VEGF and fibronectin(FN)-coated surfaces. VEGF and FN are both essential for efficient endothelial differentiation, and they exhibit a synergistic effect due to the unique structure of FN, which has a cell-adhesive site and a VEGF-binding site positioned in nanoscale proximity. However, naturally-derived FN has batch-to-batch variations. In addition, covalently immobilized FN has structural change that blocks the cell- adhesive ligand; physically adsorbed FN preserves the active cell-adhesive domain but does not allow precise control of surface ligand density. Therefore, cell microenvironments created with FN are not tightly controlled, hampering consistent production of endothelial cells from stem cells. This problem can be addressed by using well-controlled synthetic materials that recapitulate the essential molecular structure underlying the synergistic effect of VEGF and FN in regulating endothelial differentiation. The objective of this application is to develop synthetic materials having the essential structural characteristics underlying the synergistic effect of VEGF and FN and to use these materials to guide endothelial differentiation of human iPSC-derived hemangioblasts. Our central hypothesis is that a cell-adhesive peptide and a VEGF-mimetic peptide fused to a pair of heterodimerizing coiled-coils, respectively, can be brought into nanoscale proximity through coiled-coil self- assembly and the materials functionalized with the heterodimer, together with soluble factors, will create well- controlled cell microenvironments for efficient and reproducible endothelial differentiation of iPSC-derived hemangioblasts. The specific aims are: (1) design, synthesize, characterize, and immobilize the polypeptides that self-assemble to present a cell-adhesive peptide and a VEGF-mimetic peptide in nanoscale proximity; (2) examine endothelial differentiation of human iPSC-derived hemangioblasts on the polypeptide-functionalized substrates. Successful completion of this project will result in well-controlled, biomimetic cell microenvironments for efficient and robust endothelial differentiation of iPSC-derived hemangioblasts.