Human iPSC-Derived Blood Vessel Organoids for Studying Chronic Hypoxia-Induced Microvascular Dysfunction.

Microvascular dysfunction due to hypoxia is a key contributor in the pathogenesis of many disorders including cancer and retinal and cardiovascular diseases, but relevant human models are missing. Here, we present a robust 3D in vitro method with the use of human induced pluripotent stem cell-derived blood vessel organoids to analyze in vitro microvascular remodeling. We present a detailed practical pipeline combining optical tissue clearing, high-resolution immunofluorescence, and surface marker analysis to quantitatively assess hypoxia-driven changes in endothelial cells, pericytes, and the basal lamina. Exposure of these blood vessel organoids to chronic hypoxia (1% O2) for 1 week recapitulated key pathological features, including structural remodeling and a dysregulated secretome with altered vascular endothelial growth factor signaling. This approach establishes a versatile and human-relevant platform to study microvascular remodeling induced by chronic hypoxia and other pathological stimuli and their contribution to microvascular-related diseases.