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，从增强工程化的血管通畅性 血管移植和支架在促进缺血组织新生血管中的作用。但他们有限的供应 阻碍内皮细胞相关技术的成功。干细胞技术的进步提供了一种 这是解决这一问题的独特机会。特别是，内皮细胞是从人类 多能干细胞(HPSCs)，可以广泛增殖，实际上提供了无限的细胞来源。 最近在诱导PSCs(IPSCs)方面的成功提供了额外的优势 免疫兼容的自体hPSCs，并在未来实现“个性化”治疗。解决问题的关键 利用这一机会推进内皮细胞相关技术是我们引导内皮细胞 差异化。在目前使用的方法中，hPSC被分化为血管母细胞，血管母细胞既有 造血和内皮细胞潜能，随后血管母细胞分化为内皮细胞 血管内皮生长因子和纤维连接蛋白(FN)涂层表面的存在。血管内皮生长因子和纤维连接蛋白都是高效的 内皮细胞分化，由于FN的独特结构，它们表现出协同效应，FN具有 细胞黏附部位和位于纳米尺度附近的血管内皮生长因子结合部位。然而，自然衍生的FN 具有批次到批次的变化。此外，共价固定化的FN具有阻止细胞的结构变化- 粘附性配体；物理吸附的FN保留了活性细胞粘附域，但不允许精确 表面配基密度的控制。因此，用FN创造的细胞微环境不受严格控制， 阻碍干细胞持续产生内皮细胞。这个问题可以通过使用 控制良好的合成材料，概括了协同作用背后的基本分子结构 血管内皮生长因子和纤维连接蛋白对血管内皮细胞分化的调控作用此应用程序的目标是开发 具有血管内皮生长因子和血管内皮细胞生长因子协同作用的基本结构特征的合成材料 并使用这些材料来指导人IPSC来源的血管母细胞的内皮分化。我们的 中心假设是一个细胞粘附肽和一个血管内皮生长因子模拟肽融合成一对 异二聚化的螺旋线圈分别可以通过螺旋线圈的自组装而接近纳米级。 组装体和用异二聚体功能化的材料，再加上可溶性因子，将产生良好的 可控的细胞微环境促进IPSC来源的内皮细胞高效和可重复分化 血管母细胞。具体目标是：(1)多肽的设计、合成、表征和固定化 在纳米尺度上自组装成细胞粘附肽和血管内皮生长因子模拟肽；(2) 多肽功能化检测人IPSC来源的血管母细胞的内皮分化 底物。该项目的成功完成将导致控制良好的仿生细胞 IPSC来源的血管母细胞高效和稳健分化的微环境。

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.