Rpmi b27

All experiments were approved by the University of Washington Embryonic Stem Cell Research Oversight Committee (ESCRO) and conducted using the H7 (NIHhESC-10-0061) hESC line. Human pluripotent stem cells (hPSCs) were maintained and differentiated as previously described[4 (link),25 (link)]. Briefly, hESCs were cultured on Matrigel-coated plates (BD Biosciences) with mouse embryonic fibroblast-conditioned medium (MEF-CM) containing 4ng/mL basic fibroblast growth factor (bFGF). Cells were passaged using Versene-EDTA and cardiogenesis was induced with RPMI-B27 (Gibco) containing L-glutamine, Matrigel, and 10μg/mL recombinant human activin A (R&D Systems). After one day, the media was switched to RPMI-B27 containing L-glutamine and 100ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, R&D Systems), and four days later the media was aspirated and subsequently replaced every other day with RPMI-B27 containing L-glutamine. Cells typically began beating spontaneously on approximately day 12–15 post-induction. Prior to experimentation, cells were passaged using 0.05% trypsin-EDTA and replated on PEI-gelatin coated glass bottom petri-dishes as previously described[25 (link)].

Following established protocols (Cai et al., 2008 ), control, LMNA T10I, LMNA R541C hiPSCs were grown in feeder-free differentiation conditions. For efficient hepatocyte differentiation, cells were incubated in definitive endoderm media with recommended supplements (Stem Cell Technologies) for 4 days in ambient O₂ and 5% CO₂, yielding homogeneous monolayer of definitive endoderm cells. At day 5, cells were incubated with recombinant human BMP-4 (20ng/mL; Peprotech) and recombinant human FGF basic (10ng/mL; R&D Systems) for 5 days in RPMI-B27 (with insulin) in 5% O₂ and 5% CO₂, yielding hepatic progenitor cells. At day 10, cells were incubated in RPMI-B-27 (with insulin) supplemented with recombinant human HGF (20ng/mL; PeproTech) for 5 days at 5% O₂ and 5% CO₂, yielding immature HLCs. Finally, at d15, cells were incubated with HCM Hepatocyte Culture Medium (Lonza) without EGF and supplemented with recombinant human oncostatin M (20ng/mL; R&D Systems) for 7 days in ambient O₂ and 5% CO₂, yielding mature HLCs, which were collected at day 23 for subsequent ChIP, immunofluorescence, and immunoblotting.