Evaluating an Alternative Endothelial Cell Source to Vascularize Engineered Tissue Constructs

Abstract

A major translational challenge in the fields of therapeutic angiogenesis and regenerative medicine is the need to create functional microvasculature. Cell-based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are a promising, autologous, alternative cell source. The purpose of this study was to assess whether a potentially autologous endothelial cell (EC) source derived from iPSC-ECs can form the same robust, stable microvasculature as previously documented for other sources of ECs.

The endothelial lineage of iPSC-ECs was first characterized as through endothelial marker expression and compared to human umbilical vein endothelial cells (HUVECs). Similarities in endothelial markers were demonstrated and genetic expression profile analysis revealed significant genotypic similarities between the iPSC-ECs and HUVECs.

A well-established in vitro assay was utilized, in which endothelial cell-coated (iPSC-ECs or HUVECs) beads were co-embedded with fibroblasts in a 3D fibrin matrix, to assess the iPSC-ECs’ ability to form stable microvessels. iPSC-ECs exhibited a five-fold reduction in capillary network formation compared to HUVECs in this assay. Variation of cell sourcing, lot, cell density, and media formulation demonstrated no differences in iPSC-EC sprouting, eliminating these variables as the underlying cause. Despite quantitative differences, iPSC-ECs demonstrated some characteristics of mature vasculature including hollow lumen formation, pericyte recruitment and association, and deposition of basement membrane components.

To determine a cause of the in vitro sprouting attenuation, iPSC-ECs’ capillary morphogenetic mechanisms were identified through chemical inhibition of sprouting and analysis of the expression levels of key proteases. Increasing matrix density reduced sprouting, although this effect was attenuated by distributing the normal human lung fibroblasts (NHLFs) within the 3D matrix. Inhibition of both MMP- and plasmin-mediated fibrinolysis was required to completely block sprouting of both HUVECs and iPSC-ECs. Further analysis revealed MMP-9 expression and activity were significantly lower in iPSC-EC/NHLF co-cultures than in HUVEC/NHLF co-cultures, which may account for the observed deficiencies in angiogenic sprouting of the iPSC-ECs.

To investigate if the in vitro attenuation was also an in vivo phenomenon, iPSC-ECs were evaluated for their ability to form functional microvasculature in a well-established in vivo model, in which endothelial cells (iPSC-ECs or HUVECs) were co-injected with fibroblasts and a fibrin matrix into the dorsal flank of severe combined immunodeficiency (SCID) mice. Qualitatively, iPSC-ECs were capable of forming perfused vessels that inosculated with mouse vessels and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC-ECs exhibited a two-fold reduction in vessel density and a three-fold reduction in the number of perfused vessels compared to HUVECs. Further analysis revealed that the presence of the basement membrane component, type IV collagen, and the mural cell marker, alpha-smooth muscle actin, were significantly lower, roughly 25% and 33% respectively, around iPSC-EC/NHLF vasculature relative to that observed in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these findings demonstrate that a potentially autologous EC with an unlimited source, specifically iPSC-ECs, has the ability to revascularize tissue and argues for a deeper understanding of iPSC-ECs and their differences to enable the promise and potential of iPSC-ECs for clinical translation.