Wei Shen - US grants

DESCRIPTION (provided by applicant): Tissue engineering holds great promise in creating functional tissues that can replace diseased or lost tissues of human beings. Recently, consensus has been reached that three-dimensional (3D) tissue culture is superior to traditional two-dimensional (2D) cell culture in recapitulating the in vivo cell microenvironments and tissue structures. It has been found that many engineered tissues are functional only when they are developed in 3D systems. Despite the recognized importance of 3D tissue engineering and the tremendous efforts that have been made, the progress of developing large and viable 3D tissues of clinically relevant sizes has been limited. One major challenge in creating such tissue products is insufficient mass transfer in the interior region of large and a vascular constructs. Although mass transfer in large constructs prepared from preformed porous scaffolds can be enhanced in vitro through perfusion culture, insufficient mass transfer remains a problem after these constructs are implanted in vivo. On the other hand, some in situ forming hydrogels allow encapsulated endothelial cells to form capillary networks that undergo an anastomosis with the host vasculature after implantation, but hydrogel constructs without large pores cannot be perfusion-cultured so that their size is limited. Lack of methods to create large perfusable hydrogel constructs supporting in vitro endothelial capillary morphogenesis for prevascularization limits our ability to address the problem of insufficient mass transfer in 3D tissue engineering. The objective of this R21 application is to use a modular assembly approach to develop large, porous hydrogel constructs containing endothelial capillary networks and to examine postimplantation survival of the cells in these constructs. The central hypothesis of this work is that fibrin microgels having well-controlled morphology and laden with endothelial cells and other cells of interest can be modularly assembled into large, porous constructs in situ through a judiciously selected chemical reaction occurring under physiologically permissive conditions and such assembled constructs can be perfusion- cultured in vitro and develop into prevascularized, porous constructs that support high postimplantation cell survival. The Specific Aims of this project are: (1) design, fabrication, and characterization of modularly assembled large porous fibrin hydrogels laden with human umbilical vein endothelial cells (HUVECs) and hMSCs;(2) in vitro culture of large porous cell-laden constructs under perfusion and characterization of capillary morphogenesis and cell viability;(3) implantation of prevascularized porous constructs and characterization of in vivo function of the capillary networks and postimplantation survival of transplanted cells. The method proposed in this application will provide a platform to create centimeter-sized porous constructs containing capillary networks that undergo anastomosis with the host vasculature after implantation and allow interstitial flow of body fluids. Successful accomplishment of this project will address the problem of insufficient mass transfer that hampers creation of functional 3D tissue products of clinically relevant sizes. (End of Abstract)

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.