C-MSC Isolation and Culture

Mouse C-MSCs were obtained from Dr. Yaoliang Tang at Augusta University, which were isolated from the hearts of 2- to 3-month-old Notch1^flox mice (The Jackson Laboratory, stock number: 006951) according to the procedure as previously described (Ju et al., 2018). The isolated cells were purified using a mouse hematopoietic lineage depletion cocktail kit (Stemcell Technologies) and Sca-1 magnetic beads (MiltenyiBiotec Inc.) with magnetic activated cell sorting (MACS). These cells expressed the mesenchymal cell surface makers CD105, CD44, and CD140 by flow cytometric analyses (Ju et al., 2018). Cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 55 nM β-mercaptoethanol, and 1% MEM non-essential amino acid. Before EV collection, culture medium was switched to medium supplemented with exosome-deleted FBS (Gibco) for 48 h. C-MSCs isolated from Notch1^flox mice were designated as C-MSCs^{Notch1 FF}.