Vitronectin recombinant human protein vtn n

The CADASIL iPSC lines were established from skin biopsies of two CADASIL patients carrying NOTCH3 variants Arg153C and Cys224Try, respectively, as reported in our previous study (Kelleher et al., 2019 (link)). The five control iPSC clones were from three healthy individuals with two iPSC clones (02C3 and 02C9) reported by Kelleher et al. (2019) (link), two clones (SW171a and SW174a) reported by Wood et al. (2020) (link), Woods et al. (2020) (link), and OX1-19 line reported by Jarosz-Griffiths et al. (2019) (link). Prior to differentiation, all iPSCs were cultured in Vitronectin Recombinant Human Protein (VTN-N) (ThermoFisher, A14700, Loughborough, UK) pre-coated six-well plates with 2 mL TeSR-E8 medium (STEMCELL Technologies, 05990, Cambridge, UK) at 37^°C with 5% CO₂, except for the OX1-19 iPSCs that were cultured on Matrigel (Corning, 354277, Deeside, UK) pre-coated 6-well plates in mTeSR1 complete medium (STEMCELL Technologies, 85850, Cambridge, UK).
Primary human coronary arterial endothelial cells (HCAECs) (PromoCell, C-12222, Heidelberg, Germany) were cultured in six-well plates (Corning, 3516) with 2 mL Endothelial Cell Growth Medium (MV2, PromoCell, C-22121, Heidelberg, Germany) and maintained at 37^°C incubator with 5% CO₂.

Human cervix carcinoma HeLa cells (American Type Culture Collection) and human embryonic kidney HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific; Cat. No.: 41966029) supplemented with 5% fetal bovine serum (FBS). The generation, characterization and culture conditions of the human iPSCs used in this study (LUMC0020iCTRL) were detailed elsewhere (24 (link),45 (link)). In brief, the iPSCs were kept in Essential 8 Medium (E8; Thermo Fisher Scientific; Cat. No.: A1517001) supplemented with 25 U ml⁻¹ penicillin and 25 μg ml⁻¹ of streptomycin (Thermo Fisher Scientific; Cat. No.: 15140122). Vitronectin Recombinant Human Protein (VTN-N; Thermo Fisher Scientific; Cat. No.: A14700) was applied for coating all the vessels used for iPSC culturing. The different cell types were tested for the absence of mycoplasma contamination and were cultured at 37ºC in humidified-air atmospheres with 5% CO₂ (human iPSCs) or 10% CO₂ (HeLa and HEK293T cells).