2.2.1. hiPSC-CM Differentiation and Maturation

Healthy hiPSC lines SFC-854 and OX1-19 were cultured in mTeSR medium on Matrigel-coated plates and were dissociated using ReLeSR at 90-100% confluency. After 3 passages, the cells were then transferred onto Matrigel–coated 12 well plates for differentiation. The hiPSC-CM differentiation was carried out according to the procedure described in a previous report [17]. In brief, cells were cultured and expanded to 90% cell confluence and then treated for 2 days with 6 μmol/L CHIR99201 in differentiation medium (RPMI medium supplemented with 1% B-27 supplement minus insulin). On day 3, cells were treated with 2.5 mmol/L Wnt-C59 to inhibit the Wnt signalling pathway. The purification medium was applied on day 11 and 13 by changing of the medium to no-glucose RPMI with 1% B27 minus insulin and 5 mmol/L sodium lactate. hiPSC-CM maturation was initiated from day 16 to day 20 with maturation medium (MM): DMEM containing 5 mmol/L glucose supplemented with 0.4 mmol/L oleic acid conjugated to BSA, 50 nmol/L insulin, 10% fetal calf serum inactive, 1% glutamine, and 1% penicillin–streptomycin (P/S). To achieve a high glucose environment, hiPSC-CM was treated with 5.5, 11, and 25 mmol/L glucose for 2 days.