Mtesr e8 media

Dermal fibroblasts from a patient with MFS (Coriell Institute, GM21943) were reprogrammed into human induced pluripotent stem cells (iPSCs), and the mutation was corrected by CRISPR-Cas9 to generate an isogenic control.^{26 (link)} Control and Marfan iPSCs were grown on Vitronectin XF (STEMCELL Technologies) and maintained in mTeSR E8 media (STEMCELL Technologies). They were then differentiated into neural crest SMCs using the methods described previously.^{27 (link),28 (link)} After differentiation, the resulting SMCs were maintained in DMEM supplemented with 10% fetal bovine serum for at least 2 weeks before seeding onto UniFlex Culture Plates (FlexCell International Corporation). After 4 days, the SMCs were stretched for 48 hours using a Cyclic Stress Unit (FX5000 Tension System, FlexCell International Corporation), using a cyclic sine wave (60 cycles/min) and 10% elongation.

Naïve hPSC were maintained on irradiated mouse embryonic fibroblasts (MEF) in PDLGX medium prepared as following: N2B27 supplemented with 1μM PD032590, 10ng/ml human LIF (both from Cambridge Stem Cell Institute facility), 2μM Gö6983 (Tocris Bio-Techne, Cat. 2285), and 2μM XAV939 (Tocris Bio-Techne, Cat. 3748), as described previously (Rostovskaya et al., 2019 ). N2B27 basal medium was prepared as following: Neurobasal (Cat. 21103049, ThermoFisher Scientific) and DMEM/F12 (Cat. 31331093, ThermoFisher Scientific) in the ratio 1:1; 0.5% N2 (Cat. 17202048, ThermoFisher Scientific), 1% B27 (Cat. 17504044, ThermoFisher Scientific), 2mM L-glutamine (Cat. 25030024, ThermoFisher Scientific), 100μM 2-mercaptoethanol (Cat. M7522, Sigma-Aldrich). Geltrex (A1413302, ThermoFisher Scientific) was added at a concentration 0.5μl/ml to the culture medium during re-plating. Naïve hPSC were passaged using TrypLE Express (Cat. 12604021, ThermoFisher Scientific) as single cells. 10μM ROCK inhibitor (Y-27632, Cat. 688000, Millipore) was added for 24 hours after passaging.
Conventional primed H9 hPSC (WA09, WiCell) were cultured in mTeSR-E8 media (Cat. 05990, STEMCELL Technologies) (Chen et al., 2011 (link)) on Geltrex pre-coated plates and passaged using 0.5mM EDTA in PBS. All cells were cultured in a humidified incubator with 5% O₂ and 5% CO₂ at 37°C.