Supplementary Material for: Modification of a Purification and Expansion Method for Human Embryonic Stem Cell-Derived Cardiomyocytes

Objective: This study aimed to develop a simple and efficient purification method for human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) using a low-glucose culture system. In addition, we investigated whether intercellular adhesion between single hESC-CMs plays a critical role in enhancing proliferation of purified hESC-CMs. Method: hESCs were cultured in suspension to form human embryoid bodies (hEBs) from which ∼15% contracting clusters were derived after 15-20 days in culture. To purify CMs from contracting hEBs, we first manually isolated contracting clumps that were re-cultured on gelatin-coated plates with media containing a low concentration of glucose. The purified hESC-CMs were cultured at different densities to examine whether cell-cell contact enhances proliferation of hESC-CMs. Results: Purified CMs demonstrated spontaneous contraction and strongly expressed the CM-specific markers cardiac troponin T and slow myosin heavy chain. We investigated the purification efficiency by examining the expression levels of cardiac-related genes and the expression of MitoTracker Red dye. In addition, purified hESC-CMs in low-glucose culture demonstrated a 1.5-fold increase in their proliferative capacity compared to those cultured as single hESC-CMs. Conclusion: A low level of glucose is efficient in purifying hESC-CMs and intercellular adhesion between individual hESC-CMs plays a critical role in enhancing hESC-CM proliferation.