Fig. 4

Functional characterization of VAL9-HEP in vitro at day 30 of differentiation. a Glycogen storage was assessed by PAS staining. Cells were incubated with insulin 10⁻⁷ M (INS) or INS 10⁻⁷ M + glucagon 10⁻⁶ M (GLC). b Cells were examined for uptake and excretion of ICG. c Differentiation of VAL9 hESCs was assessed by flow cytometry after transduction with lentivectors expressing green fluorescent protein (GFP) under the control of apolipoprotein A-II (APOAII) and cytochrome P450 3A4 (CYP3A4) promoters or EF1α promoter in control vectors. d Ureagenesis was assessed by measuring the formation of urea from NH⁴⁺ after incubation of the cells for 24 hours in the presence of NH4CL and by comparison to neonate hepatocytes. e UDP glucuronosyltransferase 1A1 (UGT1A1) activity was assessed by incubating VAL9-HEP with 15 μM β-estradiol for 24 hours and by comparison to neonate hepatocytes. f Western blot analysis showing expression of uridine 5’-diphospho-(UDP) glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) in VAL9-HEP and in neonatal hepatocytes (NH) using β-actin as loading control. g Human (h)ALB secreted to the media by VAL9-HEP after 20 and 30 days of differentiation. hALB was determined in the culture medium by ELISA. h CYP activity levels in VAL9-HEP were determined in cells exposed to 25 μM rifampicin and were compared to the control cells. Cells were incubated for 24 hours with a cocktail of specific substrates as previously described [55]. Activity values are expressed as pmol of the corresponding metabolite formed per minute and per million cells