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.
Total RNA and miRNA were isolated from cells using an miRNeasy kit (Qiagen) with the optional column RNAse-free DNase treatment, according to the manufacturer’s instructions. 200 nanograms to one microgram of RNA was reverse transcribed into cDNA using random hexamers with Superscript III Reverse Transcriptase (Invitrogen). RNA was extracted at each stage, biotin labeled, and hybridized to either Affymetrix GeneChip Human Gene 1.0 ST or miRNA 2.0 arrays. For methylation analysis, gDNA was extracted from the same 27 samples before undergoing bisulfite conversion. Bisulfite converted DNA was then amplified and purified prior to overnight hybridization to Illumina’s Infinium HumanMethylation 450 BeadChips. Next day staining of hybridized arrays produced methylation-dependent differential fluorescence that was detected via an Illumina iScan array scanner.
Biotin labeling was performed using the Affymetrix FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol.
The labeled, fragmented DNA was hybridized to the Affymetrix GeneChip miRNA 2.0 Array for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.
After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus.
Sample name: H1 T0 microRNA expression data from the H1 embryonic stem cell line profiled at T0.
The data were analyzed with the Robust Multiarray Average (RMA) using the Affymetrix Expression Console.