Cell culture
All cell lines were maintained at 37 °C in a 5% CO2incubator. HEK293 wild type cells were maintained in DMEM supplemented
with 10% (v/v ) foetal calf serum (FCS) and integrin α4
over-expressing cells were maintained in DMEM supplemented with 10%
(v/v ) FCS and 500 μg mL-1 geneticin.
Human induced pluripotent stem cells (iPSCs) (Cell line GM23720, NIGMS
collection from the Coriell institute for medical research) were
maintained on growth factor reduced (GFR) matrigel-coated plates in
mTeSR™ Plus media (STEMCELL Technologies). Cells were passaged using
ReLeSR™ (STEMCELL Technologies) and re-plated as small clumps of cells
at a dilution of 1:10 to 1:20. For SMC differentiation, iPSCs were
dissociated with Accutase and plated on GFR Matrigel at a density of
2.5x104 cells cm-2 in ROCK inhibitor
(Y-27632, 10 µM)-supplemented mTeSR™ Plus media for 24 hours. Media was
replaced with STEMdiff™ MIM (STEMCELL Technologies) for 72 hours, with
media replaced every 24 hours. After 72 hours, the MIM was replaced with
SMC Induction medium consisting of STEMdiff™ APEL-2 medium (STEMCELL
Technologies) supplemented with 50 ng ml-1 VEGF and 25
ng ml-1 BMP4 for 4 days, with media replaced after 2
days. On day 8, cells were dissociated using Accutase and plated on
collagen IV (30 µg ml-1 coated wells) in Smooth Muscle
Cell Growth Medium 2 (SMGM2 (Promocell) supplemented with 10 ng
ml-1 PDGF-BB, 2 ng ml-1 TGFβ, 0.5 ng
ml-1 EGF, 2 ng ml-1 bFGF, 5
µg/ml-1 insulin and 0.05 ml/ml FCS for a further 14
days. Experimentation was conducted on cells between day 32 and day 40.