Immunoevasive Engineered Living Blood Vessels

项目摘要 项目调查人员最近的创新为快速评估建立了一个重要的新框架， 和可规模生产的工程活血管。值得注意的是，我们设计了新的协议， 多重基因组编辑以产生人多能干细胞（hPSC），其中HLA-A、-B和-C是 选择性消融，消除HLA II类分子，和多种致耐受性因子，包括HLA-G，PD-L1， 表达CD 47。血管平滑肌细胞（SMC）和内皮细胞（EC）来源于这些细胞， 使用我们先前报道的化学定义的分化方案，PSC被保护免于 在体外和体内的同种免疫排斥。此外，我们还开发了新的工程方法 制造机械坚固的、独立的、可折叠的胶原片和相关的制造工具， 大规模生产工程活血管。在这个提议中，我们假设免疫回避剂 使用“低免疫原性”细胞和平面细胞外基质可以有效和快速地制造血管 基质（ECM）支架的定义组成，内容，和微架构。在这个过程中， 将评价多种致耐受性策略。在本提案中，我们打算： (1)定义工程化活血管的形态和结构重塑反应 设计用于模拟天然血管壁微观结构的替代物。工程船将是 通过将原代人血管壁细胞接种在由胶原纤维组成的ECM片上来制造， 一种胶原蛋白-弹性蛋白多层复合物。生物力学特性将根据微观结构进行调整， 以及在模拟生理条件下定义的生化和功能反应。船只将 植入免疫缺陷SRG大鼠，并定义表型稳定性和重塑反应。 (2)产生“低免疫原性”血管平滑肌细胞和内皮细胞， 免疫排斥EC和SMC将衍生自通过以下方法产生的低免疫原性hPSC： 多重基因组编辑和确定的生物学特性，包括分化效率，功能性， 缺乏HLA蛋白和表达致耐受性因子。血管生成潜能和血管网络 将在体外和体内评估形成。将使用体外T细胞、NK细胞和NK细胞评价同种异体反应性。 细胞和巨噬细胞免疫测定，以及在含有人免疫系统组分的小鼠中。 (3)表征免疫逃避的表型稳定性、免疫原性和重塑应答。 人造血管将产生由低免疫原性细胞组成的工程化血管， 并表征了相关的生物力学和生物化学性质。我们将确定这些能力 在免疫缺陷SRG大鼠体内植入后，血管维持表型稳定性。在最后阶段 在这些研究中，我们将确定从低免疫原性SMC和EC工程化的血管的能力， 在用人类免疫系统的元素重建的SRG大鼠中避免免疫排斥。

Project Summary Recent innovations by project investigators have established an important new framework for the rapid and scalable production of engineered living blood vessels. Notably, we have designed new protocols for multiplex genome editing to generate human pluripotent stem cells (hPSCs) in which HLA-A, -B, and -C were selectively ablated, HLA class II molecules eliminated, and multiple tolerogenic factors, including HLA-G, PD-L1, and CD47 expressed. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) derived from these PSCs, using our previously reported chemically defined differentiation protocols, were protected from alloimmune rejection in vitro and in vivo. Further, we have developed new engineering approaches for the fabrication of mechanically robust, free-standing, ultrathin collagen sheets and related manufacturing tools for the scalable production of engineered living blood vessels. In this proposal, we postulate that immunoevasive blood vessels can be efficiently and rapidly manufactured using ‘hypoimmunogenic’ cells and planar extracellular matrix (ECM) scaffolds of defined composition, content, and microarchitecture. In the process, the efficacy of a variety of tolerogenic strategies will be evaluated. In this proposal we intend to: (1) Define the morphological and structural remodeling responses of an engineered living blood vessel substitute designed to mimic the microstructure of the native vessel wall. Engineered vessels will be fabricated by seeding primary human vascular wall cells on ultrathin ECM sheets consisting of collagen fibers or a collagen-elastin multilamellar composite. Biomechanical properties will be tuned in response to microstructure, and both biochemical and functional responses defined under simulated physiological conditions. Vessels will be implanted into immunodeficient SRG rats and both phenotypic stability and remodeling responses defined. (2) Generate ‘hypoimmunogenic’ vascular smooth muscle cells and endothelial cells that evade immunological rejection. ECs and SMCs will be derived from hypoimmunogenic hPSCs generated by multiplex genome editing and biological properties determined, including differentiation efficiency, functionality, absence of HLA proteins, and expression of tolerogenic factors. Angiogenic potential and vessel network formation will be assessed in vitro and in vivo. Alloreactivity will be evaluated using an in vitro panel of T cell, NK cell, and macrophage immunoassays, as well as in mice containing human immune system components. (3) Characterize the phenotypic stability, immunogenicity, and remodeling responses of immunoevasive engineered living blood vessels. Engineered vessels comprised of hypoimmunogenic cells will be produced and related biomechanical and biochemical properties characterized. We will determine the capacity of these vessels to maintain phenotypic stability after in vivo implantation in immunodeficient SRG rats. In the final phase of these studies, we will determine the ability of vessels engineered from hypoimmunogenic SMCs and ECs to evade immunological rejection in SRG rats reconstituted with elements of a human immune system.