Engineering Functional Capillary Networks.

Abstract

A major translational challenge in the fields of therapeutic angiogenesis and tissue engineering is the ability to form functional networks of blood vessels. Cell-based strategies to promote neovascularization have led to the consensus that co-delivery of endothelial cells (ECs) with a supporting stromal cell type is the most effective approach. However, the choice of stromal cells has varied across studies, and their impact on the functional qualities of the capillaries produced has not been examined. Our lab has developed methods to form interconnected networks of pericyte-invested capillaries both in vitro in a 3D cell culture model and in vivo. However, if the engineered vessels contain ECs that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this thesis was to determine if stromal cells of different origins yield capillaries with different functional properties, in complementary in vitro and in vivo models. In vitro, a fluorescent dextran tracer was used to visualize and quantify transport across the endothelium. In EC-fibroblast co-cultures, the dextran tracer penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, tracer accumulated at the vessel periphery, with very little transported across the endothelium. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), tighter control of permeability was achieved. In vivo, all conditions yielded new vessels that inosculated with mouse dorsal vasculature and perfused the implant, but there were significant functional differences, depending on the identity of the co-delivered stromal cells. EC alone and EC-fibroblast implants yielded immature capillary beds characterized by high levels of erythrocyte pooling in the surrounding matrix, while EC-MSC and EC-AdSC implants produced more mature capillaries characterized by less extravascular leakage and expression of mature pericyte markers. Injection of dextran tracer into the circulation also showed that EC-MSC and EC-AdSC implants formed vasculature with more tightly regulated permeability. These results suggest that the identity of the stromal cells is key to controlling the functional properties of engineered capillary networks.