Purification and Characterization of SANLCs

Purification of SANLCs referenced a metabolic-selection method of cardiomyocytes as previous described [2 (link), 9 (link)]. Briefly, SANLCs induced by three small molecule chemicals were performed in RPMI 1640 medium without d-glucose (11,879,020, Life Technologies) supplemented with 213 µg/ml l-ascorbic acid 2-phosphate (66,170–10-3, Sigma-Aldrich), 500 µg/ml O. sativa-derived recombinant human albumin (Sigma-Aldrich) for 2 days and then in RPMI 1640 medium without d-glucose supplemented with 213 µg/ml l-ascorbic acid 2-phosphate, 500 µg/ml O. sativa-derived recombinant human albumin, and 5 mM sodium dl-lactate (L4263, Sigma-Aldrich) for 3–4 days. Medium was changed to CDM3 medium for maintenance of SANLCs for 2 days. The markers of pacemaker cells were evaluated by analysis of RT-PCR and flow cytometry at day 32. Electrophysiological characteristics were analyzed using action potential (AP) recording at day 32.