4.5. In Vitro EB Formation and Trilineage Differentiation

The iPSC colonies cultured in feeder-free conditions were dissociated with 1 mg/mL collagenase IV. Embryoid body (EB) formation was carried out in suspension culture by culturing of iPSC aggregates with EB medium consisting of knockout Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific), 10% KnockOut serum replacement (KSR), 1× NEAA, 0.1 mM β-mercaptoethanol, and 1 mM L-glutamine in ultralow attachment plates (Corning, Corning, NY, USA) for 5 days. For spontaneous trilineage differentiation, the EB aggregates were transferred to gelatin-coated plates, cultured for an additional 10 days, and analyzed for trilineage marker expression by immunocytochemistry. The medium was changed every other day. The antibodies utilized are summarized in Supplementary Table S2.