KRIT1 heterozygous mutations are sufficient to induce a pathological phenotype in patient-derived iPSC models of Cerebral Cavernous Malformation

Cerebral Cavernous Malformation (CCM) is a neurovascular disease distinguished by clusters of leaky, mulberry-like blood vessels. KRIT1 biallelic loss-of-function mutations in endothelial cells are known to trigger brain cavernomas, however, human pre-clinical models are needed to unveil the importance of germline KRIT1 heterozygous mutations in CCM pathogenesis. We generated three iPSCs from CCM patients with hereditary KRIT1 heterozygous mutations. Patient-derived vascularized organoids exhibited intricate and abnormal vascular structures with cavernoma-like morphology, and iPSCs-derived endothelial cells displayed phenotypic abnormalities at the junctional and transcriptional level. Upon injection into brain explants, CCM-endothelial cells integrated into the normal vasculature and created vascular anomalies. Lastly, transcriptional analysis showed that the endothelial progenitor marker PEG3 was highly expressed in iPSCs-derived CCM endothelial cells and further confirmed in familial and sporadic cavernoma biopsies. Overall, our study enlightens the molecular consequence of KRIT1 heterozygous mutations in endothelial cells and the potential implications in cavernoma pathogenesis. Overall design: CCM patient-derived and one healthy control iPSCs were differentiated towards endothelial cells using a ETV2 tetracycline inducible vector. Furthermore, the same cells (only patient CCM01 and Healthy control) were co-cultured with brain explants ex vivo.