Single-cell characterization of neovascularization using hiPSC-derived endothelial cells in a 3D microenvironment

The formation of vascular structures is fundamental forin vitrotissue engineering. Vascularization can enable the nutrient supply within larger structures and increase transplantation efficiency, which are currently limiting factors in organoid research. We differentiated human induced pluripotent stem cells toward endothelial cells in 3D suspension culture. To investigatein vitroneovascularization and various 3D microenvironmental approaches, we designed a comprehensive single-cell transcriptomic study. Time-resolved single-cell transcriptomics of the endothelial and co-evolving mural cells gave insights into cell type development, stability, and plasticity. Transfer to a 3D hydrogel microenvironment induced neovascularization and facilitated tracing of sprouting, coalescing, and tubulogenic endothelial cells states. During maturation, we monitored two pericyte subtypes evolving of mural cells. Profiling cell-cell interactions between pericytes and endothelial cells confirmedin vivoangiogenic signaling and emphasized new cytokine signals during tubulogenesis. Our data, analyses, and results provide anin vitroroadmap to guide vascularization in future tissue engineering.