Cardiomyocyte support medium

hiPSC-CMs were obtained from commercial products (JK Med, China) and could be observed to be spontaneously beating under a microscope (Supplementary Information File: Section S3). After washing in 1× dulbecco’s phosphate buffered saline (DPBS, Gibco, USA) twice for 5 min, hiPSC-CMs were digested with EDTA Solution (Stemcell Technologies, Canada) for 10 min, followed by the addition of cardiomyocyte support medium (Stemcell) to terminate digestion, centrifugation to remove the supernatant, and dilution of the hiPSC-CM suspensions with cardiomyocyte support medium.
The primary HUVECs were obtained from commercial products (JK Med), and passages 4–7 were used for the experiments (Supplementary Information File: Section S3). HUVECs were cultured in Endothelial Cell growth Medium-2 (EGM-2, CC-3162, Lonza, Switzerland) supplemented with 1% penicillin–streptomycin solution (Hyclone, China). HUVECs were grown to 90% confluence before use in cell culture flasks. After washing in 1× DPBS twice for 5 min, the HUVECs were removed from the cell culture flask using 0.25% trypsin solution (Saimike, China). Then, dulbecco’s modified eagle medium: F-12 (DMEM/F12, Hyclone) with 10% fetal bovine serum (FBS, Hyclone) was added to terminate digestion, the samples were centrifuged to remove the supernatant, and HUVEC suspensions were diluted with EGM-2.

Cardiomyocytes were re-plated between days 14–16 as standard using the cardiomyocyte dissociation medium from STEMCELL Technologies and following manufacturer’s guidelines (STEMCELL Technologies). Cells were plated onto new dishes coated with GFR Matrigel (Corning), Fibronectin (Sigma) or Geltrex (Thermo Fisher Scientific) at a seeding density of 3x10⁵ cells/cm², or as indicated, in cardiomyocyte support medium (STEMCELL Technologies) supplemented with 10 μM Y-27632 (Tocris). After 24 h, the media was changed to the appropriate insulin containing media. Cells were re-plated on other days for specific assays following the same protocol.