Antibiotic antimycotic solution

After preculture for 48 h, fibroblasts were exposed to 5 μg/mL or 50 μg/mL of poly I:C (polyinosinic-polycytidylic acid sodium salt) (4287/10; R&D Systems, Minneapolis, MN, USA). Poly I:C is a mixture of long and short reagents. During preculture and poly I:C exposure, the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; 043–30085; FUJIFILM Wako Pure Chemical Industries) containing 10% FBS (SH30396.03; Cytiva) and 1% penicillin-streptomycin-amphotericin B suspension (×100) (Antibiotic-Antimycotic Solution) (161–23181; FUJIFILM Wako Pure Chemical Industries). Cells were cultured at 37°C in 5% CO₂.

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.

Pluripotent stem cell-enriched human dental pulp-derived cells (hDPSCs) were provided by JCR Pharmaceuticals (Hyogo, Japan). The cells were cultured in Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) at 37 °C in an atmosphere containing 5% CO₂.

MSCs derived from rat bone marrow were isolated and expanded as previously described [26 (link)]. For the experiments on dogs, healthy donor Beagle dogs were anesthetized using thiopental and isoflurane, and 1.0 mL of bone marrow fluid was collected. The CD271⁺ MSCs were enriched and cultivated using the MSC Research Tool Box-CD271 (LNGFR) containing CD271 (LNGFR)-PE and anti-PE microbeads for cell separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), as previously reported [27 (link)]. Human DPSCs were provided by JCR Pharmaceuticals (Hyogo, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) at 37 °C in a 5% CO₂ atmosphere.

Spherical clusters of iPSCs re-suspended in DMEM/F12 (Gibco) containing 20% KSR (Gibco), 2 mM GlutaMAX (Gibco), 100 μM non-essential amino acid (NEAA) (Gibco), 100 μM 2-mercaptoethanol (Sigma), and a 0.5% antibiotic-antimycotic solution (FUJIFILM Wako) were transferred to Petri dishes. After an 8-day floating culture, spontaneously formed EBs were transferred to ECL (Millipore)-coated plates and incubated for another 8 days.