NOTCH3 signal activation by its extracellular domain accumulation in an iPSC line newly established from a CADASIL patient.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cerebral small-vessel disease caused by NOTCH3 mutations, yet its pathogenic mechanisms remain incompletely understood due to limited disease models. The NOTCH3 R544C mutation is a prevalent hotspot in East Asian populations, but patient-derived iPSC models are lacking. Here, we generated an iPSC line from peripheral blood mononuclear cells of a middle-aged CADASIL patient carrying a heterozygous NOTCH3 c.1630C > T (p.Arg544Cys, R544C) mutation using a Sendai virus (SeV)-based reprogramming approach. The iPSCs exhibited typical morphology, normal 46, XY karyotype, expressed pluripotency markers (OCT4, NANOG, TRA-1-60, SSEA-4), and cleared SeV vectors after passaging. They differentiated into derivatives of all three germ layers, and STR analysis confirmed donor identity. Functionally, CADASIL iPSCs showed abnormal accumulation of the NOTCH3 extracellular domain (NOTCH3ECD) with unchanged NOTCH3 full-length and intracellular domain levels, and upregulation of canonical downstream genes HEY1, NRARP, and HES1, indicating activation of the NOTCH3 signaling pathway. This study establishes and characterizes a NOTCH3 R544C patient-derived iPSC line, providing a valuable model for investigating CADASIL pathogenesis and potential therapeutic strategies, with novel insights into early NOTCH3ECD accumulation and pathway activation.