Nonessential amino acid solution

Hepatic stellate cells (HSCs) were isolated from 35 ± 5-week-old C57BL/6 female mice. Briefly, to obtain the HSCs, the livers were digested with liver perfusion medium (Thermo Fisher Scientific) and liver digestive medium (Thermo Fisher Scientific). Non-parenchymal cells from the digested cells were fractionated using 11% HistoDenz (Sigma-Aldrich, St. Louis, MO, USA) at 2500 rpm for 20 min. After isolation, mouse HSCs were cultured on collagen type I-coated 12-well plates (AGC Techno Glass Co., Ltd., Haibara, Japan) in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin-glutamine (Thermo Fisher Scientific). After 6 h of culture, cells were washed with PBS, the medium was changed, and ONO-1301 (0.1 μM) or DMSO (control group) was added to each well. The medium was changed 24 h later. After 72 h, HSCs were harvested, and the mRNA expression levels of genes encoding activated HSC factors (α-smooth muscle actin [Acta2], type I collagen alpha 1 [Col1a1], type III collagen alpha 1 [Col3a1]), and quiescent HSC factors (cytoglobin [Ctgb] and Hedgehog interacting protein [Hhip]) were evaluated using real-time PCR (Supplementary Table 1).

A total of 50 ml of OPC-PM is prepared mixing 30 ml of DMEM/F12 with 1 ml of B27, 0.5 ml of N2, 0.5 ml of GlutaMax (2 mM), 0.5 ml of Penicillin-streptomycin (50 U/ml), 0.5 ml of Non-essential amino acid solution (0.1 mM), 0.5 ml of sodium pyruvate (1 mM; all purchased from Thermo Fisher Scientific), 10 μl of platelet-derived growth factor-AA (PDGF-AA, 20 ng/ml, stock concentration: 100 μg/ml; PeproTech), and 20 μl of bFGF (20 ng/ml, stock concentration: 50 μg/ml; PeproTech). DMEM/F12 is added to a final volume of 50 ml, filter with a bottle-top filter (0.22 μm) and store at 4°C. We suggest to use complete media within 2 weeks.

After 7–8 days of differentiation, the insert chamber and the Cell-Disc were transferred to an 8-well plate (two chambers or two Cell-Discs per well) placed in a C-Dish (IonOptix, Milton, MA) for EPS. EPS (1 Hz, 1 ~ 6 ms, 20 V/25 mm) was applied to the cells in the C-Dish using a C-Pace 100 pulse generator (IonOptix). DMEM containing 2% CS supplemented with final two-fold concentrations of amino acids, achieved by adding 50X amino acid solution (Thermo Fisher, #11130036) and 100X non-essential amino acid solution (Thermo Fisher, #11140050), was used during the EPS treatments, as previously reported^{1 (link)}. A total of 4, 8, 16 or 24 h of EPS was applied and the cells were then harvested for analyses. In the experiments with a total of 16 or 24 h of EPS, 1 h intervals were set between the 8 h EPS sessions to prevent unwanted detachment of vigorously contracting myotubes from the substratum of the insert-chamber^{7 (link)}.

To evaluate the material cytocompatibility, human fetal lung fibroblasts (MRC-5; Merck, USA) were chosen. The cells were regularly passaged using trypsinization and maintained at the exponential phase of growth. Cultivation media consisted of Minimal essential medium (MEM; Merck, USA) supplemented with 2 mM l-glutamine, 1 % (v/v) non-essential amino acid solution, and 10 % (v/v) fetal bovine serum (all from Thermo Fisher Scientific, USA). The MRC-5 cells were kept in a cell culture incubator at 37 °C, an atmosphere with 5 % CO₂ and 95 % humidity.

PBMC were cultured (2 x 10⁶ viable cells/mL) in complete media containing Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, St. Loius, MO, USA) with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA), 23.8mM sodium bicarbonate (Fisher Scientific, Waltham, MA, USA), 7.5 mM HEPES (Amresco, Framingham, MA, USA), 170 μM Penicillin G (Tokyo Chemical Industry, Portland, OR, USA), 137 μM Streptomycin (Sigma, Burlington, MA, USA), 50 μM β-mercaptoethanol (Sigma, Burlington, MA, USA), 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), essential amino acid solution (Thermo Fisher Scientific, Waltham, MA, USA), non-essential amino acid solution (Thermo Fisher Scientific, Waltham, MA, USA), 500 ng/mL R848 (Invivogen, San Diego, CA, USA) and 5 ng/mL rIL-2 (R&D, Minneapolis, MN, USA) for 7–9 days at 37°C in 5% CO₂ [63 (link), 81 (link)]. Conditioned medium supernatants were harvested and evaluated for total and rHA-specific IgG abundance by ELISA starting at a 1:5 dilution. Frequency of B cells amongst total viable PBMC was assessed by CD19 surface labeling and flow cytometry analysis.

EGR-G01 ES cells were generated in the Ikawa Lab [28 (link)] and cultured in KnockOut DMEM (108297–018, Thermo Fisher Scientific) supplemented with 1% Penicillin-Streptomycin- Glutamine, 55 μM 2-mercaptoethanol, 1% Non-Essential Amino Acid Solution (11140–050, Thermo Fisher Scientific), 1% Sodium Pyruvate (11360–070, Thermo Fisher Scientific), 30 μM Adenosine (A4036, Sigma- Aldrich, St. Louis, MO, USA), 30 μM Guanosine (G6264, Sigma-Aldrich), 30 μM Cytidine (C4654, Sigma-Aldrich), 30 μM Uridine (U3003, Sigma-Aldrich), 10 μM Thymidine (T1895, Sigma-Aldrich), 100 U/ml mouse LIF, and 20% FCS (51650–500, Biowest, Nuaillé, France).

EB formation was performed as previously described^{35 (link)}. iPSCs were scraped off, dissociated in primate ES medium, and distributed into low-attachment six-well plates (Corning) for floating culture with EB medium (Knockout Dulbecco’s Modified Eagle Medium, 20% KSR, 0.1 mM non-essential amino acid solution, 2 mM l-glutamine, 500 U/mL P/S, and 0.5 mM 2-mercaptoethanol; all reagents were obtained from Thermo Fisher Scientific). On day 8, the EBs were transferred into gelatin-coated six-well plates and cultured with EB medium for another 8 days.