2 mercaptoethanol

SFEBq-based differentiation was performed as previously described [19 (link), 20 (link)]. iPSCs were dissociated into single cells and quickly re-aggregated in DFK 5% medium (DMEM/F-12 Ham medium (Sigma-Aldrich) containing KnockOut™ Serum Replacement (Thermo Fisher Scientific), NEAA (Thermo Fisher Scientific), 2-mercaptoethanol (Nacalai), GlutaMAX (Thermo Fisher Scientific), SB-431542, Dorsomorphin (Tocris, Minneapolis, MN, USA), and Y-27632 (Tocris)) (9000 cells/well) using a Nunclon Sphera Microplates 96U-Well Plate (Thermo Fisher Scientific). The medium was changed every 4 days until day 12, after that we changed to neurobasal medium supplemented with B27 supplement (Thermo Fisher Scientific) and GlutaMAX (Thermo Fisher Scientific) for further neural differentiation. Neurospheres were fixed in 4% paraformaldehyde for 30 min at room temperature followed by dehydration with 30% sucrose overnight and then cryosectioned at 20 μm with Leica CM1860 Cryostat (Leica Biosystems, Wetzlar, Germany).

MIN6 cells, a mouse-derived beta cell line [10 (link)], were cultured at 37°C with 5% CO₂ in DMEM (Sigma–Aldrich, St. Louis, MO, USA) containing 450 mg/dl glucose supplemented with 10% FBS (Sigma–Aldrich), 100 U/ml penicillin (Nacalai Tesque, Kyoto, Japan), 100 μg/ml streptomycin (Nacalai Tesque), and 100 μM 2-mercaptoethanol (Nacalai Tesque).
409B2 cells, a human iPS cell line derived from a healthy individual, were purchased from RIKEN Bioresource Centre Cell Bank (Ibaraki, Japan). 409B2 cells were cultured over the Mitomycin C-treated SNL feeder cells at 37°C with 5% CO₂ in Primate ES medium (ReproCELL, Kanagawa, Japan) supplemented with 4 ng/ml recombinant human basic fibroblast growth factor (Wako, Osaka, Japan) and 500 U/ml penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). At 70–80% confluence, 409B2 cells were induced to insulin-producing cells using the differentiation protocol described previously [16 (link)]. Briefly, cells were first differentiated into endodermal cells expressing sex-determining region Y-box 17 (stage 1, 4 days), then into pancreatic progenitor cells expressing pancreatic and duodenal homeobox-1 (stage 2, 6 days), and finally into insulin-producing cells (stage 3, 12 days). The iPS cell study protocol was approved by the Ethics Committee of Osaka University.

iPSCs were treated with Accutase and plated on Matrigel-coated plates in StemFit+Y at 3.0 × 10⁴ cells/cm². After 24 and 48 h, the media was replaced with PECM and PECM+0.3 µg/mL Dox, respectively. After an additional 48 h, the pre-differentiated iPSCs were treated with Accutase and re-plated into new Matrigel-coated microplates or hydrogels in 2% HS/αMEM supplemented with 200 µM 2-mercaptoethanol (Nacalai), 4.5 g/L glucose (Invitrogen), 10 µg/mL insulin (Wako; Richmond, VA, USA), SB431542 (Wako) and 3 µM Y-27632. After two days, the medium was replaced with 2% HS/αMEM+1 µg/mL Dox and changed every other day. To induce further maturation of myotubes, doxycycline was removed from the media and the cells were cultured in 2% HS/αMEM for 3 weeks. The medium was replaced every other day.

253G1 line (passage number: P32 – P38), 454E2 line (P34), and RIKEN-2A line (P19) (RIKEN Cell Bank, Ibaraki, Japan.) were used in this study. All experiments were performed in accordance with ethical guidelines of our university and the cell bank. Undifferentiated hiPS were maintained on SNL 76/7 cells (ECACC, Salisbury, UK) as a feeder layer, as previously described18 (link). The undifferentiated hiPS cells were cultured in Dulbecco’s modified Eagle medium/F12 (DMEM/F12, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% (v/v) knockout serum replacement (KSR, Life Technologies, Carlsbad, CA, USA), 0.1 mM nonessential amino acid (NEAA, Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Nacalai Tesque, Kyoto, Japan), and 5 ng/mL basic fibroblast growth factor (bFGF, Wako Pure Chemical Industries, Osaka, Japan) in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. The hiPS cells were subcultured every three days using CTK solution (0.25% (v/v) trypsin and 0.1 mg/mL collagenase (Nitta Gelatin Inc., Osaka, Japan) in PBS (-) supplemented with 20% (v/v) KSR and 1-mM calcium chloride). The details of the automated hiPS culture are described in Fig. 2. The manual culture was conducted by an expert with five years of experience with hiPS culture.

Myogenic differentiation was performed according to a previous protocol,³⁴ (link) with some modifications. MyoD-iPSCs were dissociated into single cells by incubation with 0.5× TrypLE Select, and single cells were seeded on growth factor reduced Matrigel (Corning)-coated dishes without feeder cells. Matrigel was diluted 1:100 with primate ESC medium (ReproCELL), and the culture medium was changed to primate ESC medium with 10 μM Y-27632 but without bFGF. After 24 h, Dox (1 μg/mL; LKT Laboratories) was added to the culture medium. After an additional 24 h, the culture medium was changed to differentiation medium, composed of alpha minimum essential medium (α-MEM) (Nacalai Tesque) with 5% KnockOut Serum Replacement (KSR) (Gibco), a 0.5% antibiotic-antimycotic solution (FUJIFILM Wako), and 200 μM 2-mercaptoethanol (Nacalai Tesque). After an additional 5 days, the culture medium was changed to DMEM (high glucose; Nacalai Tesque) with 2% horse serum (Sigma), a 0.5% antibiotic-antimycotic solution, 2 mM GlutaMAX (Gibco), and 200 μM 2-mercaptoethanol.

Splenocytes were isolated and cultured as previously described [9 (link),30 (link)]. Briefly, splenocytes (4 × 105 cells/well) were cultured for 48 h on flat-bottomed 96 well plates (Corning Costar, Cambridge, MA, USA) coated with 5 μg/mL anti-CD3ε monoclonal antibody (mAb) in 200 μL Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 50 μM 2-mercaptoethanol (Nacalai Tesque, Inc., Kyoto, Japan) and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (SAFC Biosciences, Lenexa, KS, USA) and 1% (v/v) antibiotic-antimycotic solution (10,000 U/mL penicillin, 10,000 μg/mL streptomycin and 25 μg/mL amphotericin B; Life Technologies, Gaithersburg, MD, USA). The cultures were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. After 48 h, IFN-γ, IL-4, and IL-10 levels in culture supernatants were evaluated using cytokine-specific enzyme-linked immunosorbent assays (ELISAs) that are commercially available from BD Biosciences.