Pluripotent stem cells were differentiated as previously described (Kurian et al, 2012). Briefly, undifferentiated human ES/iPS cells were freshly split on to matrigel-coated plates, making sure the sub-colonies were of small size (~300-500 cells /colony). Cells were differentiated using the chemically defined mesodermal induction media (MIM) (DMEM:F12, 15mg/ml stem cell grade BSA (MP biomedicals), 17.5µg/ml Human Insulin, (SAFC bioscience), 275 µg/ml Human holo transferrin (Sigma Aldrich), 20ng/ml bFGF (Hymanzyme), 50ng/ml Human VEGF-165 aa (Humanzyme), 25ng/ml human BMP4 (Stemgent), 450µM monothioglycerol (Sigma Aldrich), 2.25mM each L-Glutamine and Nonn-essential amino acids (Invitrogen).
Human ES cells, H1 (WA1, WiCell), (passage 25-40) were cultured in chemically defined hES/hiPS growth media, mTeSR on growth factor reduced matrigel (BD biosciences) coated plates. Briefly, 70-80% confluent hES/hiPS cells were treated with dispase (Invitrogen) for 7 minutes at 37¡C and the colonies were dispersed to small clusters and lifted carefully using a 5ml glass pipette, at a ratio of ~1:6. Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Promocell and cultivated in EBM medium supplemented with EGM singlequots (Lonza). iPS/ES-derived endothelial cells were cultivated in EGM-2 bullet kit (Lonza). All the endothelial cells were grown on collagen I coated plates (BD biosciences). All cell lines were maintained in an incubator (37¡C, 5% CO2) with media changes every day (hES/hiPS) or every second day (HUVEC).
genomic DNA was extracted and purified from samples using Qiagen DNeasy Kit according to standard instructions
Cy5 and Cy3
Standard Illumina Protocol
bisulphite converted DNA was amplified, fragmented and hybridised to Illumina 450K Infinium Methylation Arrays using standard Illumina protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting