Fig. 3

Characterization of iHeps in vitro. a Immunofluorescence analysis of ALB and AAT in iHeps. More than 90 % of iHeps efficiently expressed both ALB and AAT at day 9. b Secretion of ALB increased during the hepatogenic induction period as measured by ELISA. c The cumulative urea amounts gradually increased in the iHeps culture system during the hepatogenic induction period. d Gene expression profile analysis of ADSCs, iHeps, and rHeps cells by cDNA microarray. Hierarchical clustering showed that iHeps were grouped together with rHeps. e Analysis of basic hepatic function in iHeps, including PAS staining, ac-LDL, and ICG uptake. f mRNA levels of CYP genes were determined by quantitative PCR in iHeps and rHeps before inducer treatment. Data normalized to the level of GAPDH. g mRNA levels of the induced CYP enzymes were measured by quantitative PCR. CYP1A1and CYP1A2 was induced by 3-methylcholanthrene. CYP2A1 and CYP3A1 were induced by phenobarbital. CYP2E1 was induced by acetone. Fold induction in iHeps and rHeps was normalized to levels in cells without inducer treatment, respectively. h CYP metabolic activity in iHeps. CYP enzymes were induced in iHeps for 48 hours. Fresh rHeps were directly used as a positive control. The metabolic products of phenacetin (acetaminophen, assay for CYP1A2 activities), coumarin (7-hydroxycoumarin, assay for CYP2A1 activities), and chlorzoxazone (6-hydroxychlorzoxazone, assay for CYP2E1 activities) were determined by liquid chromatography–tandem mass spectrometry. Scale bar: 100 μm (a, e). AAT alpha-1-antitrypsin, ac-LDL acetylated low-density lipoprotein, ADSC adipose-derived stem cell, ALB albumin, CYP cytochrome P, ICG indocyanine green, iHep induced functional hepatocyte, PAS Periodic acid–Schiff, rHep primary rat hepatocyte, UD undetectable