4.1. Isolation and Culture of CardiopoieticAF and hiPSC Cells

AF samples were obtained from 8 women undergoing amniocentesis for prenatal diagnosis at 16–17 weeks of pregnancy after written informed consent, in accordance with the Declaration of Helsinki. All samples presented normal diploid male karyotypes, as evidenced by cytogenetic investigation. The mean (±SD) maternal age at amniocentesis was 37.9 ± 2.6 years. The study was approved by the local ethics committee and all experiments were performed in accordance with relevant guidelines and regulations.

^{Cardiopoietic}AF cells were isolated as previously described on the basis of the phenotypic pattern [2] cultured in Iscove’s modified Dulbecco’s medium (IMDM, Lonza Group Ltd., Basel, Switzerland), supplemented with 20% fetal bovine serum (FBS, GE Healthcare HyClone Defined Fetal Bovine Serum, Buckinghamshire, UK), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, (all Sigma-Aldrich, Saint Louis, MO, USA), and 5 ng/mL basic fibroblast growth factor (bFGF, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37 °C with 5% humidified CO₂.

hiPSCs [49] were cultured on a monolayer of irradiated murine fibroblasts (Thermo Fischer Scientific, Waltham, MA, USA) in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 20% KnockOut Serum Replacement (KO) (Thermo Fischer Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Thermo Fischer Scientific, Waltham, MA, USA), 2 mM L-glutamine (Corning, New York, NY, USA), 5 ng/mL basic fibroblast growth factor (bFGF) (Thermo Fischer Scientific, Waltham, MA, USA), and 1% MEM nonessential amino acid (Corning, New York, NY, USA).