Esgro supplement

Primary Sca1⁺Bmi1⁺CD45⁻ and PDGFRα⁺ mesenchymal-like cells were sorted after Langendorff digestion and expanded in Iscove's modified Dulbecco's medium (IMDM, ThermoFisher, 12440053) containing 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin and 2 mM l-glutamine (all from Invitrogen), 10³ units ESGRO Supplement (Millipore, ESG1106), 10 ng/ml EGF (epidermal growth factor; Sigma, SRP3196) and 20 ng/ml FGF (fibroblast growth factor; Peprotech, 100-18B) (37 °C, 3% O₂, 5% CO₂). Primary adult cardiac endothelial cells (CD31⁺) were obtained with the Neonatal Cardiac Endothelial Cell Isolation Kit (Miltenyi, 130-104-183). CD31⁺ primary cells and the 1g11 endothelial cell line [39 (link)] were expanded in VascuLife VEGF Endothelial Medium Complete Kit (Lifeline Cell Technology, LL-0003) (37 °C, 21% O₂, 5% CO₂). Primary cardiac cells were used for the experiments at passage ≤9.

Fifty thousand skin fibroblasts were plated per well on a 6-well plate two days before infection, and medium containing 5:5:3 mix of lentiviral vectors expressing KOS, hc-Myc, and hKlf4 was applied to cells. The medium was replaced with fresh fibroblast medium 24 h after transfection, and cells were cultured for one week with medium exchange every other day. The medium was then replaced with mouse ES cell culture medium (Dulbecco modified Eagle medium [DMEM] with 10% fetal calf serum [FBS], 100 IU/mL penicillin G, 100 μg/ml streptomycin, 0.25 μg/mL amphotericin B, 2 mM L-glutamine, 0.10 mM nonessential amino acids and 0.05 mM β-mercaptoethanol, 1x mouse recombinant Leukemia Inhibitory Factor (LIF) (ESGRO® Supplement, Millipore), 1x MEK/GSK-3 inhibitor supplement (MEK/GSK-3 Inhibitor Supplement, Millipore). The putative iPS cell colonies were identified and chosen using morphological selection criteria. Mouse iPS cell colonies were dissociated with TVP solution (PBS with 1% chicken serum, 0.025% trypsin, and 1.27 mM EDTA) and passage onto a 0.1% gelatin-coated dish containing mouse ES cell culture medium, which was replaced every other day.