Follistatin

Recombinant human TGF-β1, activin A, activin B, activin AB, follistatin, and neutralizing monoclonal anti-TGF-β1,-β2,-β3 antibody (#MAB1835) were all from R&D Systems (Wiesbaden, Germany). The ALK4/5/7 inhibitor SB431542 was purchased from Calbiochem/Merck (Darmstadt, Germany) and dissolved in dimethylsulfoxide.

PMA was purchased from BIAFFIN GMBH & CO KG. TGFβ1, EGF, Follistatin, and R-spondin 1 were purchased from R&D systems. Wortmannin and Pentagastrin were purchased from Sigma. Noggin was purchased from PeproTech. SB431542 was obtained from Wako. To generate Wnt3A-conditional medium, L-Wnt-3A cells (ATCC, CRL2647) were cultured with the DMEM-High Glucose medium (Invitrogen), which contains 10% Knockout Serum Replacement, 1% Non-Essential Amino Acid Solution, Penicillin-Streptomycin, 2 mM L-Glutamine, and 100 μM β-mercaptoethanol, for 24 hr, and then the conditioned medium was collected.

Hepatocytes were generated from hPSCs by a growth factor‐based differentiation protocol described in our previous protocol [15]. After 20 days of differentiation, the cells were harvested by using a serial 2× TrypLE Express treatment and further dissociated into single cells by passing them through a 40‐μm cell strainer. These single cells were then seeded at 2.5 × 10⁵ cells per well in a collagen I (50 μg/ml, Bio Laboratories, Singapore, http://www.biolab.com.sg, catalog no. 354236)‐coated dishes. Attachment and recovery were promoted by seeding them in step IV differentiation medium with hepatocyte growth factor (R&D Systems, catalog no. 294‐HGN‐005), Follistatin (R&D Systems, catalog no. FS‐288), Oncostatin (R&D Systems, catalog no. 295‐OM‐010), and Y‐27632 (Rock Inhibitor) to prevent anoikis in the freshly harvested hPSC‐HEPs. The next day, medium was changed to Williams E medium (Sigma‐Aldrich, catalog no. W1878) without serum, and cells were serum‐starved overnight before nutraceutical treatments. Nutraceuticals quercetin (Sigma‐Aldrich, catalog no. Q4951) and genistein (Sigma‐Aldrich, catalog no. G6649) were administered at a single dose of 10 μM. Hepatocytes in this work were derived from H9‐ESCs. In hepatic characterization, primary human hepatocytes (PHHs) and HUH7 cells were used as positive controls, and HeLa cells were used as negative controls.

Enzyme linked immunosorbent assay was used for the quantitative measurement of activin-A and follistatin (R&D systems, Minneapolis, USA) in serum and liver homogenates samples. All samples were processed in duplicate and according the manufacturer’s instructions. The optical density of the plates was measured within 10 min using a plate reader at 450 nm and correction at 560 nm as recommended by the manufacturer.
The lowest detection limit of activin-A by the used kit was 3.7 pg/ml and the upper limit was 1,500 pg/ml as reported by the manufacturer. The intra-assay and inter-assay precisions of the kit were 4.3 and 5.8%, respectively. The kit could cross react by 0.2 and 0.45% with inhibin-A and activin-AB, respectively. The detection range of the follistatin kit was 250–16,000 pg/ml and the minimum detectable dose was 83 pg/ml.

HDLECs were purchased from Lonza (Basel, Switzerland). The cells were maintained in EBM ^TM-2MV Bullet Kit (cc-3202, Lonza) and used for study after extensive characterization. HEK-Blue^TM TGF-β cells (HEK293 cell-derived TGF-β responsive reporter cells) were purchased from InvivoGen (San Diego, CA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (Nacalai Tesque: Kyoto, Japan) supplemented with 10% Fetal Bovine Serum (FBS, SIGMA: St. Louis, MO, USA), 100 units/mL Penicillin-100 μg/mL Streptomycin (Nacalai Tesque). TGF-β2, TNF-α, and Activin A were purchased from Peprotech (Rocky Hill, NJ, USA). Follistatin was purchased from R&D Systems (Minneapolis, MN, USA). SB431542 and Y27632 were obtained from WAKO Chemicals (Saitama, Japan). Concentrations of TGF-β2 used were determined empirically, based on their ability to alter expression of various endothelial and mesenchymal markers.