H1 hesc

H1 hESCs (Wicell) were cultured on Biomatrix in feeder-free culture conditions with mouse embryonic fibroblast-conditioned media (MEF-CM) (Tan et al., submitted for publication). Briefly, human embryonic stem medium [80% Dulbecco's modified Eagle's medium-Ham's F-12 medium, 20% Knockout serum replacement (Invitrogen), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids and 4 ng/ml human basic fibroblast growth factor (bFGF)] was incubated with mitomycin C-inactivated MEFs overnight, with an additional 4 ng/ml of bFGF before hESC feeding. Cultures were passaged every 5–7 days, before they became confluent. Differentiation was induced via the addition of specific factors into MEF-CM, along with the removal of bFGF. For trophoblast induction, cultures were incubated in MEF-CM with 100 ng/ml of recombinant BMP4 (R&D Systems) and 20 μM of FGFR-inhibitor SU5402 (Calbiochem) for 1–5 days (30 (link)). For retinoid differentiation, cultures were incubated in MEF-CM with 10 μM of synthetic retinoid EC23 (Reinnervate) for 1–5 days (31 (link)).

H1 hESCs were purchased from WiCell in Wisconsin (WA01) and cultured in TeSR medium in Matrigel-coated 10-cm plates. Cells were grown at 5% O₂ tension to better mimic the conditions inside the human body and reduce oxidative damage as a result of normoxic conditions. To passage the cells, and just prior to collection of RNA and DNA, cells were gently treated with 2 µg/mL Dispase mixed with Dulbecco's Modified Eagle Medium/F12, washed with phosphate buffered saline (PBS), and scraped off the plate using a glass pipette. DNA and RNA were then isolated with standard phenol chloroform and Trizol methods.