High-throughput differentiation of human blood vessel organoids reveals overlapping and distinct functions of the cerebral cavernous malformation proteins
Authors: Skowronek Dariush , Pilz Robin A. , Saenko Valeriia V. , Mellinger Lara , Singer Debora , Ribback Silvia , Weise Anja , Claaßen Kevin , Büttner Christian , Brockmann Emily M. , Hübner Christian A. , Aung Thiha , Haerteis Silke , Bekeschus Sander , Ekici Arif B. , Felbor Ute , Rath Matthias 
Published: December 2024

ABSTRACTCerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in theCCM1,CCM2, orCCM3gene. Despite a better understanding of CCM pathogenesis in recent years, it is still unclear whyCCM3mutations often lead to a more aggressive phenotype thanCCM1orCCM2variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with aCCM1,CCM2, orCCM3knockout, single-cell RNA sequencing, and high-content imaging, we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions, inactivation ofCCM1,CCM2, orCCM3also led to specific gene expression patterns in neuronal, mesenchymal, and endothelial cell populations, respectively. Taking advantage of the different fluorescent labels of the hiPSCs, we could also visualize the abnormal expansion ofCCM1andCCM3knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast,CCM2knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant inCCM2and to decode the molecular and cellular heterogeneity in CCM disease. Finally, the ability to differentiate blood vessel organoids in a 96-well format will further facilitate their use in drug discovery and other biomedical research studies.STATEMENTS AND DECLARATIONSConflicts of interest statementThe authors declare no competing interests. The here described protocol for high-throughput organoid synthesis has been filed as a patent application at the European Patent Office (Process number: EP24213596.0)Author contribution statementMR, DSk, and UF designed the study. DSk, VS, LM, and RAP performed most of the functional experiments. SH and TA performed the CAM assays. SR performed the immunohistochemical stainings. SB, DSi, DSk, and VS performed the confocal microscopy and high-content imaging analyses. AE, CB, and EMB performed the scRNA sequencing analysis. AW and CAH performed and analyzed the karyotyping of the hiPSC clones. DSk, RAP, VS, KC, MR, and SB analyzed the data. DSk, VS, LM, and MR prepared figures. All authors contributed to the interpretation of the results. DSk, RAP, VS, and MR drafted the manuscript, and all authors contributed to writing.Ethics statementThis study does not involve human participants or animal subjects.Availability of data and materialsAll relevant data are published within the paper and the supplementary files. ScRNA sequencing data can be accessed through the Gene Expression Omnibus (GEO) database (record number: GSE276497).