Immunoevasive Engineered Living Blood Vessels

点击翻译按钮获取中文摘要

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.