Directed Differentiation of iPICs for Implantation

Stage 7 medium was based on a previous report [7 (link)] with our original modifications. Cells were cultured in the MCDB 131 medium with 1% P/S, 2% fat-free bovine serum albumin (FUJIFILM Wako), 20 mM glucose, 1.5 g/L NaHCO₃, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5iII or the alternative candidates, 1 μM T₃, 10 μM ZnSO₄ (Merck Millipore), 1.4 IU/mL heparin sodium salt (Nacalai Tesque, Kyoto, Japan), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck Chemicals, Houston, TX, USA), 1 μM PD-166866, 3 μM TR06141363 (Multi-kinase inhibitor, Takeda Pharmaceutical Company), and 10 μM Y-27632, for 4 days. To generate iPICs for implantation, cells were dissociated and re-sized in an Elplasia 6-well microwell plate (Corning Incorporated, Corning, NY, USA) or a gas-permeable microwell culture bag (Toyo Seikan Group Holdings, Ltd., Yokohama, Japan) [20 (link)] and cultured in the stage 7 medium.