Figure 5

Secretion pattern of several hepatic proteins by hiPSC-EB-HLCs. Conditioned media from hiPSC-EB-HLCs were collected after 48 hours from the completion of the differentiation protocol for both conditions with and without endothelial cells. (a) Albumin, (b) fibrinogen and (c) Alpha Fetoprotein (AFP) were detected in the medium and (d) intracellular Urea was detected. Differences in secretion between the conditions with endothelial cells were statistically significant with respect to the condition without endothelial cells for the Albumin and AFP. There was not statistically significant difference between the two experimental conditions for the Fibrinogen and Urea intracellular concentration. Undifferentiated hiPSCs were used as negative control, and human primary hepatocyte as positive control. The results are representative of at least three independent experiments. Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; Detoxification property analysis of the differentiated HLCs. (e) The ammonium metabolism assay conducted on a period over 24-hour for both conditions with and without endothelial cells showed a higher ability of ammonium clearance for the hiPSC-EB + EC-HLCs (about 45% from the first hour) when compared with hiPSC-EB-HLCs (about 20% from the first hour); (f) Phase II detoxification analysis through resorufin conjugation assay: The results showed a higher formation rate for the condition hiPSC-EB + EC-HLCs compared with the hiPSC-EB-HLCs, reaching similar level of the HPH used as positive control. (g–n) Cytochrome P450 (CYP450) induction analysis: Several CYP enzymes were assessed through incubation of the differentiated HLCs with specific inducers: Omeprazole for the (g) CYP1A1, and (h) CYP1A2; Rifampicin for the (i) CYP3A4, and (l) CYP3A7; and Phenobarbital for the (m) CYP2B6, and (n) CYP2C9 for a period of 72 hours. DMSO was used as control to test the basal activity of the different CYP450. Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.