Rapid Anastomosis of Endothelial Progenitor Cell–Derived Vessels with Host Vasculature Is Promoted by a High Density of Cotransplanted Fibroblasts

Abstract

To ensure survival of engineered implantable tissues thicker than approximately 2-3 mm, convection of nutrients and waste products to enhance the rate of transport will be required. Creating a network of vessels in vitro, before implantation (prevascularization), is one potential strategy to achieve this aim. In this study, we developed three-dimensional engineered vessel networks in vitro by coculture of endothelial cells (ECs) and fibroblasts in a fibrin gel for 7 days. Vessels formed by cord blood endothelial progenitor cell-derived ECs (EPC-ECs) in the presence of a high density of fibroblasts created an interconnected tubular network within 4 days, compared with 5-7 days in the presence of a low density of fibroblasts. Vessels derived from human umbilical vein ECs (HUVECs) in vitro showed similar kinetics. Implantation of the prevascularized tissues into immunecompromised mice, however, revealed a dramatic difference in the ability of EPC-ECs and HUVECs to form anastomoses with the host vasculature. Vascular beds derived from EPC-ECs were perfused within 1 day of implantation, whereas no HUVEC vessels were perfused at day 1. Further, while almost 90% of EPC-EC-derived vascular beds were perfused at day 3, only one-third of HUVEC-derived vascular beds were perfused. In both cases, a high density of fibroblasts accelerated anastomosis by 2-3 days. We conclude that both EPC-ECs and a high density of fibroblasts significantly accelerate the rate of functional anastomosis, and that prevascularizing an engineered tissue may be an effective strategy to enhance convective transport of nutrients in vivo.

Stromal Cell Identity Influences the In Vivo Functionality of Engineered Capillary Networks Formed by Co-delivery of Endothelial Cells and Stromal Cells

“…Fibrin is a naturally occurring biopolymer that acts as the provisional matrix during wound healing in the human body, and has been widely shown in the literature to support neovascularization. 22,33,35,39,40 The animal model used here has also been widely exploited in the literature to approximate wound healing and test the ability of transplanted human cells to form vasculature, 19,22,29,[33][34][35] in part because the human cells injected into SCID mice are not rejected.…”

“…41 A high density of cotransplanted NHLFs, in particular, in a prevascularized tissue construct has been shown to accelerate the rate of inosculation between host vessels and the implanted vessels. 29 Likewise, AdSCs co-delivered with ECs in collagen-based implants have been shown to enhance vascularization, and to help sustain pancreatic islets or adipocytes. 34 There are also a number of studies that have explored the utility of BMSCs co-delivered with ECs to build functional vasculature.…”

“…23 Despite the consensus of this paradigm, there is virtually no consensus with respect to the choice of cells to co-deliver with ECs. A variety of stromal cell types of mesenchymal origins have been explored, including mesenchymal stem cells from bone marrow 21,24,25 or adipose tissue, 26,27 fibroblasts from human lung, 28,29 and mouse embryos, 30 as well as smooth muscle cells. 31 For a subset of these cell types, our previous studies using in vitro models have shown that stromal cell identity underlies differences in the mechanisms by which capillaries are formed, 24,26 and in the functional properties of the resulting capillaries.…”

Induction of erythropoiesis using human vascular networks genetically engineered for controlled erythropoietin release

“…19 As expected, the presence of MSCs was critical to maintaining EPO expression, because successful ECFC engraftment is dependent on the presence of perivascular cells. 15,16,18,19,24 Indeed, in the absence of MSCs, the hematocrit increased only during the first week, but returned rapidly to basal levels 2 weeks after implantation (supplemental Figure 11).The measurement of mRNA in whole organs revealed a significant down-regulation of mEPO in the kidneys of mice that had borne implants for 5 weeks (74.1% reduction; *P Ͻ .01; Figure 3G), an indirect confirmation of exogenous hEPO functionality. Two other organs, heart and liver, showed low-to-undetectable levels of mEPO expression and no differences between control and implant-bearing mice.…”

“…19 As expected, the presence of MSCs was critical to maintaining EPO expression, because successful ECFC engraftment is dependent on the presence of perivascular cells. 15,16,18,19,24 Indeed, in the absence of MSCs, the hematocrit increased only during the first week, but returned rapidly to basal levels 2 weeks after implantation (supplemental Figure 11).…”