Aside from differentiation and sorting as described above, cells were not treated in any specific manner prior to DNA/RNA extraction.
iPSC and ESC lines were maintained in “hiPSC Media” composed of DMEM/F12 (Sigma-Aldrich, www.sigma.com) with 20% KnockOut Serum Replacement (Invitrogen, www.invitrogen.com), 1mM nonanimal L-glutamine (Sigma-Aldrich), 0.1mM Β-mercaptoethanol, and 10 ng/ml FGF2 (R&D Systems, Minneapolis, MN) on 0.1% gelatin (Sigma-Aldrich) coated plates preseeded with mitomycin C-inactivated or irradiated mouse embryonic fibroblast (MEF) feeder cells. Cells were maintained in a 5% CO2 air environment. For endodermal differentiation, cells were passed onto matrigel-coated dishes at 80% confluency. On the following day, designated “T0”, differentiation was induced by culture in media containing growth factors listed below. From T0-T6, differentiation media included 2mM l-glutamine, and 4.5x10-4 M monothioglycerol (MTG). Cells were grown in T0 media, consisting of RPMI-based serum-free medium with Chir 99021 (2ug/ml) and Activin A (100 ng/ml), for one day. On days “T1-2”, medium was changed to RPMI with BMP4 (0.5 ng/ml), FGF2 (10ng/ml), Activin A (100 ng/ml), and VEGF (10 ng/ml). On days “T3-4”, cells were cultured in SFD media(Gouon-Evans et al., 2006) with BMP4 (0.5 ng/ml), FGF2 (10ng/ml), Activin A (100 ng/ml), and VEGF (10 ng/ml). For hepatic differentiation, PSCs were differentiated as monolayer cultures as outlined above to generate definitive endoderm and then further differentiated for an additional 3 weeks in SFD-based media with ascorbic acid (50mcg/ml), monothioglycerol (4.5x10-4 M ), and the following supplements: T7-12: BMP4 (50 ng/ml), FGF2 (10 ng/ml), VEGF (10 ng/ml), EGF (10ng/ml), TGFa (20 ng/ml), HGF (100 ng/ml), and 0.1 uM Dexamethasone; T13-18: FGF2 (10 ng/ml), VEGF (10 ng/ml), EGF (10 ng/ml), HGF (100 ng/ml), Oncostatin M (20 ng/ml), Vitamin K (6 ug/ml), 1.5 uM gamma secretase inhibitor, 0.1 uM Dexamethasone, and 1% DMSO; T19-24: HGF (100 ng/ml), Oncostatin M (20 ng/ml), Vitamin K (6 ug/ml), and 0.1 uM Dexamethasone. Differentiating human PSCs were maintained in a 5% CO2, 5% O2, 90% N2 environment.
According to Illumina protocol
Cy5 and Cy3
500ng gDNA underwent bisulfite conversion of unmethylated cytosine bases to uracil, and bisulfite converted DNA was then amplified and purified
Overnight hybridization to BeadChip arrays.
Next day staining of hybridized arrays produced methylation-dependent differential fluorescence that was detected via an Illumina iScan array scanner.
Sample name: H1 T0 Gene methylation data from the H1 embryonic stem cell line profiled at T0.
Arrays were visualized and processed using the GenomeStudio software package, which produced IDAT files that were read into a MethyLumiSet using the methylumIDAT function in the methylumi R package (version 2.4.0). This object was then coerced to a MethyLumiM object and quantile-normalized by sequentially applying the lumiMethyC and lumiMethyN functions from the lumi R package (version 2.10.0). All methylation analyses were performed using the R environment for statistical computing (version 2.15.1).