Cellular and Molecular Characterization of CADASIL Using In Vitro Models of Vascular Smooth Muscle Cells and Blood Vessel Organoids

Abstract Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common inherited form of stroke, characterized by a NOTCH3 mutation on chromosome 19. Despite many genetic and functional studies, the exact mechanisms and pathologies have not been fully elucidated. Herein, the cellular and molecular characteristics of vascular smooth muscle cells (VSMCs) and constituent cells of blood vessel organoids are investigated. Fibroblasts derived from a patient with CADASIL are reprogrammed into iPSCs and differentiated into VSMCs, cerebral organoids, and blood vessel organoids. In CADASIL iPSC-derived VSMCs, NOTCH3 extracellular domain antibody is detected. In CADASIL-blood vessel organoids, significant differences in the blood vessel related parameters between CADASIL and control blood vessel organoids are found. Additionally, cerebral organoids by CADASIL-iPSCs are distinct from those by control-iPSCs. Furthermore, based on RNA sequencing, differentially expressed genes between control and CADASIL samples are associated with proliferation rate, cell cycle, and nuclear division in VSMCs and angiogenesis and vasculogenesis in blood vessel organoids. Six distinct clusters in blood vessel organoids and cell proportion differences between control and CADASIL are identified using single-cell RNA sequencing. This study highlights CADASIL VSMCs and 3D organoids may facilitate investigating molecular and cellular mechanisms underlying CADASIL pathogenesis.