RNA sequencing analysis of neural crest-derived vascular smooth muscle cells differentiated from CADASIL patient and control iPSCs.
CADASIL patient and control iPSCs were differentiated into neural crest-specific VSMCs. Neural crest progenitors were generated by culturing iPSCs on vitronectin-coated plates under defined Essential 6 medium (Gibco; A1516401) with 10 ng/mL FGF2 (PeproTech; 100-18B) and 10 μM SB431542 (Tocris; 1614) that promote neural crest specification. Cells were first passaged at day 5 and subsequently upon reaching confluence, with continued supplementation of FGF2 and SB431542, up to passage 12. After this intermediate stage, cells were dissociated and transferred into SMC differentiation medium composed of Essential 6 supplemented with 10 ng/mL PDGF-BB (PeproTech; 100-14B) and 2 ng/mL TGF-β1 (PeproTech; 100-21) for a minimum of 12 days. Neural crest-derived VSMCs reached maturity by day18, at which point total RNA was isolated from the fully differentiated iPSC-derived VSMCs for RNA-seq analysis. The induced pluripotent stem cells were cultured in Essential 8 Medium (Thermo Fisher Scientific) Total RNA from CADASIL patient and control iPSCs derived neural crest-specific VSMCs was extracted using the RNeasy Mini Kit (Qiagen). Illumina TruSeq Stranded mRNA kit, according to the manufacturer's protocol.