Blood vessel organoids derived from diabetic patients revealed impaired function based on a subpopulation of endothelial cells

Introduction
Impaired function of blood vessels can lead to cardiovascular diseases (CVDs), which is a major cause of death worldwide. The presence of both endothelial cells (ECs) and mural cells is central to the proper function of blood vessels in health and pathological changes in diseases including diabetes. Although iPSCs-derived vascular organoids (VOs) provide an appealing source for in vitro vascular disease modelling and drug testing, whether these organoids can recapitulate human vascular disease is yet to be determined.

Methods
Blood mononuclear cells from six donors with diabetes (DB) and three with non-diabetes (ND) were subjected to reprogramming into iPSCs, and subsequent differentiation to VOs. DB-VOs and ND-VOs were compared using immunohistochemistry, angiogenic array, ROS production assay, acetylated-LDL uptake assay, transmission electron microscopy, western blotting, flow cytometry and single-cell RNA sequencing. Additionally, the regenerative potential of DB-VOs vs ND-VOs was compared by assessment of blood recovery via Laser Doppler imaging in mice ischaemic hindlimb model, and organoid cells’ integration into host vasculature was tracked by Bruker fluorescent imaging.

Results
We showed diabetic derived-iPSCs-VOs represent impaired vascular function including enhanced ROS level, with higher mitochondrial content and activity, increased pro-inflammatory cytokines, and less regenerative potential in vivo. Using single-cell RNA sequencing, we identified all specialized types of vascular cells (artery, capillary, vein, lymphatic and tip cells, as well as pericytes and vSMCs) within vascular organoids, while demonstrating the dichotomy landscape of ECs and mural cells. Furthermore, we revealed basal heterogeneity within vascular organoids and demonstrated differences between diabetic and non-diabetic VOs. Of note, a subpopulation of ECs significantly enriched for ROS and oxidative phosphorylation hallmarks in DB-VOs, representing early signs of aberrant angiogenesis in diabetes. For the first time, we report that GAP43 (Neuromodulin) is expressed in ECs, and GAP43+ ECs are distinctly increased in DB-VOs. Therefore, GAP43 is possibly a biomarker for the onset and progression of diabetic-related blood vessel dysfunction.