RNF213 deficiency in human iPSC-derived vascular organoids captures key feature of moyamoya disease vasculopathy.

OBJECTIVES

This study aimed to establish a human iPSC-derived model of Moyamoya disease (MMD) by generating RNF213-knockdown vascular organoids to capture key disease-relevant vascular features.

METHODS

RNF213-knockdown iPSC lines were established using lentiviral shRNA and selected with puromycin. Knockdown efficiency was validated by qRT-PCR and Western blot. Vascular organoids were differentiated from the engineered iPSCs using the STEMdiff™ Blood Vessel Organoid Kit according to the manufacturer's protocol. Organoids were analyzed by immunofluorescence for vascular markers (CD31, COL4A1, PDGFR-β, α-SMA) and transcriptomic profiling was performed using DRUG-seq.

RESULTS

Three distinct RNF213-knockdown iPSC clones were successfully generated, exhibiting varying knockdown efficiencies. qPCR analysis demonstrated RNF213 mRNA reductions of approximately 22% (V1), 70% (V2), and 88% (V3), with corresponding decreases in protein levels confirmed by Western blot. Although V2 and V3 clones displayed more substantial molecular knockdown, organoids derived from the V1 clone manifested the most severe phenotypic abnormalities, characterized by disrupted CD31+ endothelial networks and Collagen IV+ basement membranes. Transcriptomic profiling identified 1543 differentially expressed genes, with enrichment analysis revealing significant dysregulation of angiogenesis-related pathways. This included marked upregulation of pro-angiogenic factors (CCL5, EREG) and downregulation of inhibitory factors (TNMD), effectively recapitulating key molecular features of Moyamoya disease.

CONCLUSION

We developed a novel human vascular organoid model with RNF-213 deficiency that captures key features of MMD vasculopathy, providing a valuable platform for mechanistic studies and drug screening.