Isolation and Culture of Human Cardiac Progenitor Cells

Human cardiac progenitor cells (hCPCs) were isolated from the right atrial appendage and sorted for expression of c-kit cell surface marker, as described previously (Zhang et al., 2017 (link)). Cells were used at passage 7–10 for these studies. Cells were initially cultured for 48 h at normoxic conditions (37°C, 21% O₂) then placed in medium with exosome-depleted FBS (SBI, Palo Alto, CA, United States) and continuously cultured at normoxic condition of 21% O₂ physoxic condition of 5% O₂ or hypoxic condition of 1% O₂ using a controlled C-chamber incubator (ProOx P110 O₂ Controllers, BioSperix, Parish, NY, United States). Media was refreshed every other day, retaining 20% of conditioned media. Phase-contrast images were captured using a DM IL LED microscope and MC170 HD digital camera (Leica Microsystems, Inc., Buffalo Grove, IL, United States).