Spatial localization of endothelial cells in heterotypic spheroids influences Notch signaling.

Cell-based therapeutic approaches are an exciting strategy to replenish compromised endothelial cell (EC) populations that contribute to impaired vasculogenesis. Co-cultures of ECs and mesenchymal stromal cells (MSCs) can enhance neovascularization over ECs alone, but the efficacy of cells is limited by rapid cell death upon implantation. Co-culture spheroids exhibit improved survival compared with monodisperse cells, yet little is known about the influence of spatial regulation of ECs within co-culture spheroids. We hypothesized that EC sprouting from co-culture spheroids is a function of EC spatial localization. We formed co-culture spheroids containing ECs and MSCs in two formats: ECs uniformly distributed throughout the spheroid (i.e., mixed) or seeded on the perimeter of the MSC core (i.e., shell). Qualitative observations suggested increased vasculogenesis for mixed co-culture spheroids compared with shell conformations as early as day 3, yet quantitative metrics did not reveal significant differences in network formation between these 3D structures. Notch3 expression demonstrated significant increases in cell-cell communication in mixed conformations compared with shell counterparts. Furthermore, knockdown of Notch3 in MSCs abrogated the vasculogenic potential of mixed spheroids, supporting its role in promoting EC-MSC contacts. This study highlights the direct impact of EC-MSC contacts on sprouting and provides insight to improve the quality of network formation. KEY MESSAGES: • Endothelial cell (EC) localization can be controlled in co-culture EC-MSC spheroids. • Mixed spheroids exhibit consistent networks compared to shell counterparts. • Differences in NOTCH3 were observed between mixed and shell spheroids. • NOTCH3 may be a necessary target for improved vasculogenic potential.