Nanoscale Assembly of Bioactive Ligands to Enhance Endothelial Differentiation

描述（由申请人提供）：内皮细胞具有重要的生物医学应用，从增强工程血管移植物和支架的通畅到促进缺血组织的新生血管。但它们有限的可用性阻碍了内皮细胞相关技术的成功。干细胞技术的进步为解决这一问题提供了一个独特的机会。特别是，内皮细胞来源于人类多能干细胞（hPSCs），它可以广泛增殖，实际上提供了无限的细胞来源。最近成功制造诱导性造血干细胞（iPSCs）为提供免疫兼容的自体造血干细胞和未来的“个性化”治疗提供了额外的优势。利用这一机会推进内皮细胞相关技术的关键是我们引导内皮细胞分化的能力。在目前使用的方法中，将造血干细胞分化为具有造血和内皮潜能的成血管细胞，然后在VEGF和纤维连接蛋白（FN）包被的表面下将成血管细胞分化为内皮细胞。VEGF和FN对于内皮细胞的有效分化都是必不可少的，由于FN的独特结构，它们表现出协同效应，其具有细胞粘附位点和VEGF结合位点位于纳米级附近。然而，自然衍生的FN具有批次到批次的变化。此外，共价固定的FN具有结构变化，可阻断细胞粘附配体；物理吸附的FN保留了活性的细胞粘附结构域，但不允许精确控制表面配体密度。因此，用FN创建的细胞微环境没有受到严格控制，阻碍了干细胞内皮细胞的持续生成。这个问题可以通过使用控制良好的合成材料来解决，这些合成材料概括了VEGF和FN在调节内皮细胞分化中的协同作用的基本分子结构。本申请的目的是开发具有VEGF和FN协同作用的基本结构特征的合成材料，并使用这些材料指导人类ipsc衍生的成血管细胞的内皮分化。我们的中心假设是，细胞粘附肽和vegf模拟肽分别融合到一对异源二聚体的线圈上，可以通过线圈自组装达到纳米级的接近，与异源二聚体功能化的材料，以及可溶性因子，将为ipsc衍生的血管母细胞的有效和可复制的内皮分化创造良好的细胞微环境。具体目标是：(1)设计、合成、表征和固定化多肽，这些多肽可以自组装成纳米级接近的细胞粘附肽和vegf模拟肽；(2)研究多肽功能化底物对人ipsc衍生的成血管细胞内皮分化的影响。该项目的成功完成将为ipsc衍生的成血管细胞的高效和稳健的内皮分化提供良好控制的仿生细胞微环境。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The proposed project aims to engineer rationally designed, well-controlled synthetic cell microenvironments to guide efficient and reproducible endothelial differentiation of hemangioblasts derived from human induced pluripotent stem cells. Such derived endothelial cells will have important biomedical applications ranging from enhancing the patency of engineered vascular grafts and stents to promoting neovascularization in ischemic tissues.