Forskolin

Human fetal ovary pieces were digested with an enzyme mixture containing 1 mg/mL Collagenase IV (Invitrogen), 0.5 mg/mL Hyaluronidase (Sigma-Aldrich) and 20 U/mL DNAse I (Sigma-Aldrich) for 30–45 min at 37°C, followed by vigorous pipetting to break up cell clumps. The single cell suspension was passed through a 40 μm nylon mesh strainer (Corning). To form the aggregates, 30,000 fetal ovary cells were combined with 5,000 sorted hPGCLCs per well of an ultra-low 96-well V-bottom plate, in aRB27 medium supplemented with RevitaCell. 3 days later, the aggregates were embedded in 1.5% low-melting agarose droplets in aRB27 medium supplemented with 100 ng/mL SCF, 5 μM forskolin (Biogems) and 150 μM ascorbic acid (Sigma-Aldrich). Medium changes were performed every 3 days and on day 17 the medium was switched back to aRB27 basal medium. On day 25, the reconstituted aggregates were isolated by breaking up the agarose droplets and fixed with 4% PFA at 4°C o/n for immunofluorescence analysis.

In order to obtain SC-ASCs, FBS-cultured ASCs were cultured in the presence of neurogenic factors as previously described [20 (link), 22 (link)]. In particular, ASCs were cultured in complete medium with 1 mM β-mercaptoethanol (SigmaAldrich) for 24 h and with 35 ng/mL all-trans-retinoic acid (Peprotech) for the subsequent 72 h. Afterward, the medium was supplemented with 5 ng/mL PDFG (Peprotech), 10 ng/mL bFGF (Peprotech), 14 µM forskolin (Biogems), and 252 ng/mL GGF-2 (Peprotech) and changed every 72 h for an overall period of 14 days.

Human iPSCs (CGT-RCiB10, Cell and Gene Therapy Catapult) were differentiated into iHEPs essentially as described^{44 (link)}, except for the Matrigel (Corning) sandwich and addition of forskolin (5 μM; BioGems) from day 17. On day 19 hepatocyte growth factor was removed and taurine was added (58.4 mg/L; Sigma) and the cells are placed in normoxia. On day 25 the iHEPs were treated +/−20 nM staurosporine (Generon) for 2 h followed by addition of the MRP2 transport substrate precursor CDFDA (10 µM; Sigma-Aldrich) and NucBlue Live Cell ReadyProbes Reagent (Thermo Fisher Scientific) and incubated for a further 15 min at 37 °C. CDFDA is cleaved by intracellular esterases to yield fluorescent 5-(and-6)-carboxy-2′,7′–dichlorofluorescein (CDF; green) which is secreted into the bile canaliculi by MRP2. Cell nuclei and accumulation of CDF (green) in bile canaliculi were captured in images taken on an LSM 880 confocal microscope (ZEISS). Images were analysed using Fiji^{45 (link)}. Images were first processed using global thresholding to filter out noise. Then, size-exclusion filtering was applied to remove particles <5 µm² to select biologically relevant signals to determine integrated density of CDF staining in the bile canaliculi. The statistical significance of experimental data was assessed by Student’s test.