Knockout sr xenofree cts ksr

Hepatic induction of iPSCs was accomplished as described previously with minor modifications [39 –41 (link)]. In brief, single cells were generated after Accutase incubation (Sigma-Aldrich) for 6–8 minutes. Cells were resuspended in mTeSR-1 and transferred to Matrigel-precoated cell culture plates. After 24 hours, culture medium was changed for the next 3 days to DMEM/F12 supplemented with 100 ng/ml recombinant Activin-A (R&D Systems), 100 ng/ml fibroblast growth factor-2 (FGF2, Peprotech), 50 ng/ml recombinant human Wnt3a (R&D Systems). Concentration of KnockOut SR Xenofree CTS (KSR, Gibco) was 0%, 0.2% and 2.0% for the first, second and third day, respectively. For the next 8 days, cells were cultivated in DMEM/F12 supplemented with 10% KSR, 1mM NEAA, 1mM L-Glutamine, 1% dimethyl sulfoxide (DMSO, Sigma-Aldrich), and 100 ng/ml hepatocyte growth factor (HGF, Peprotech). Finally, cells were grown for 3 days in 10% KSR, 1mmol/L NEAA, 1mM L-Glutamine, and 0.1μM dexamethasone (Sigma-Aldrich).

For cholangiocyte progenitor-like cell differentiation a protocol used before for hepatocyte-like cells was slightly modified^{18 (link)}. Briefly, iPSCs clusters instead of single cells were seeded to Matrigel-precoated wells in order to achieve an almost confluent cell layer at the next day. The medium was changed daily for the next three days to DMEM/F12 supplemented with 100 ng/ml recombinant Activin-A (R&D Systems), 100 ng/ml fibroblast growth factor-2 (FGF2, Peprotech), 50 ng/ml recombinant human Wnt3a (R&D Systems; day 1 and 2), 1 mM l-Glutamine (l-Glu, Sigma). KnockOut SR Xenofree CTS (KSR, Gibco) was added at 0.2% (day 2) and 2% (day 3). For the next 8 days (days 4–11), cells were cultivated in DMEM/F12 supplemented with 10% KSR, 1 mM NEAA, 1 mM L-Glu, 1% dimethyl sulfoxide (DMSO, Sigma), and 100 ng/ml hepatocyte growth factor (HGF, Peprotech). The maturation step, was prolonged up to day 15. 0.1 μM dexamethasone medium was added on day 12 to the medium (DMEM/F12, 10% KSR, 1 mM NEAA, 1 mM L-Glu, 1% DMSO) without any further medium changes at day 14 and day 15.