The differentiation of the iPSCs into vascular cells was carried out as described previously (Sone et al., 2007; Tatsumi et al., 2011). Briefly, undifferentiated human iPSCs were harvested and transferred to a collagen I-coated dish after adjusting the colonies to an appropriate size. On the second day of incubation, the culture medium was replaced with Primate ES medium without basic fibroblast growth factor (bFGF), supplemented with N2 supplement (Invitrogen)/B27 supplement (Invitrogen) and 6-bromoindirubin-3'-oxime (BIO, 5 μM, SIGMA). Thereafter, the cells were incubated for another 3 days, at which time the culture medium was replaced with StemPro-34 SFM (Invitrogen) supplemented with recombinant human vascular endothelial growth factor (VEGF, 50 ng/ml, PeproTech). After another 3–5 days of incubation, FLK1 (+) VE-cadherin (+) and FLK1 (+) VE-cadherin (-) cells were sorted individually using a FACSAria flow cytometer (BD). Sorted FLK1 (+) VE-cadherin (+) cells were confirmed to remain VE-cadherin positive during the subsequent cell culture and analyses. On the other hand, the sorted FLK1 (+) VE-cadherin (-) cells were differentiated into vascular smooth muscle cells after an additional 18-22 days of differentiation on collagen I-coated dishes supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB, 20 ng/ml, PeproTech).
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the miRNeasy Purification kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).
Label
Cy3
Label protocol
Manufature's protocol
Hybridization protocol
Manufature's protocol
Scan protocol
Slides were scanned on the G2565CA Microarray Scanner System (Agilent).
