Embryonic stem cell fetal bovine serum

Cardiac derived stem like cells (CTSCs) are isolated from the hearts of C57BL/6 Mice (Jackson) as described previously [2 ]. Briefly, hearts were minced in small pieces and digested in collagenase at 150U mg/ml (Worthington Bio Corp, Lakewood, NJ) for 2 h at 37 °C. After myocyte removal at low-speed centrifugation, the cells are then passed through 100 μm and 50 μm filters (BD biosciences CA), centrifuged at 1200 rpm for 5 min, with resuspension of the pellet in CTSC media consisting of DMEM-F12 (Gibco), 10% Embryonic Stem Cell Fetal Bovine Serum (Gibco), 1% Penicillin-Streptomycin-Glutamine (PSG) (Gibco), 1x Insulin-Transferrin-Selenium (Gibco), Recombinant Human-EGF 10 ng/mL (Peprotech), Recombinant Human-FGF 10 ng/mL (Peprotech), and Leukemia Inhibitory Factor (Millipore Sigma), and incubated in a 37 °C 5% CO₂ incubator. Supernatant was replated 24hrs later in a dish to remove fast adhering cells. CTSCs are infected with Lv-PGK1-LIN28a (CTSC-LIN) and green fluorescent protein (GFP) lentivirus (CTSC-GFP) to create stable cell lines kept between passages 15–22 to minimize variation. Additional details provided in the online data supplement.

All CTSCs were isolated from the hearts of C57BL/6 Mice (Jackson). Hearts were subjected to novel tissue digestion method, and separation of various cellular populations by plating, and replating of supernatant to remove fast adhering cells. CTSC Growth Media composition was similar to previously published¹⁹ and included DMEM‐F12 (Gibco), 10% Embryonic Stem Cell Fetal Bovine Serum (Gibco), 1% Penicillin‐Streptomycin‐Glutamine (PSG) (Gibco), 1x Insulin‐Transferrin‐Selenium (Gibco), Recombinant Human‐EGF 10 ng/mL (Peprotech), Recombinant Human‐FGF 10 ng/mL (Peprotech), and Leukemia Inhibitory Factor (Millipore Sigma), and incubated in a 37°C 5% CO₂ incubator. Routine cell culturing passaging included dissociation of cells using 0.25% Trypsin EDTA (Gibco) and centrifugation for 5 minutes at 1500 RPM. Pelleted cells were resuspended in CTSC Growth media and counted using a hemocytometer and plated. For all subsequent experiments, all three CTSCs types were kept between passages 10 and 15.