M dex

Activation of the HRind cells was done by addition of 10⁻⁷ M Dex (Sigma, Aldrich) and of the U2OS-DRGFP cells by ISceI infection, using adenovirus (provided by F. Graham), similarly to (18 (link)). Analyzing GFP-positive cells out of the mCherry-positive cells in flow cytometric analysis was performed using FACSAria Cell-Sorting System (Becton Dickinson), similarly to Sartori et al. (33 (link)). For each compound, the background was set in the absence of DSB induction and reduced using the same gate. Sample analysis was done in FlowJo program.

After setting the optimal conditions for DE induction, we aimed to generate stepwise functional hepatocyte-like cell. The gut tube and hepatocyte specifications were initiated using RPMI medium supplemented with 2% KOSR, 10 ng/ml FGF4, and 10 ng/ml HGF (R&D Systems). Three days later, RPMI was replaced with the enriched minimum essential medium (MEM, Sigma-Aldrich) supplemented with 1% BSA, 10 ng/ml FGF4, and 10 ng/ml HGF. Three days later, hepatocyte maturation was induced using complete hepatocyte culture medium (HCM, SingleQuots, Lonza, USA) with all supplements according to the manufacturer's recommendations, containing 10 ng/ml FGF4, 10 ng/ml HGF, 10 ng/ml OSM, and 10⁻⁷ M Dex (Sigma-Aldrich). The hepatocyte maturation was proceeded for nine days as previously reported by Ishkitiev et al. [21 (link), 22 (link)]. The differentiation protocol was performed for a total of 22 days, and the media were refreshed every 2-3 days.