Bovine mesenchymal stem cells (bMSCs) were isolated, cultured, and characterized based on generally accepted criteria [69 (link),70 (link)] as in [71 (link)]. The bMSC-SV40-hTERT cell line immortalization was achieved by introducing simian virus 40 large T antigen (SV40T, a kind gift from Prof. Sara Selig) by transient transfection, and the human telomerase gene hTERT [72 ] by retrovirus-mediated transduction. The cells were grown in low-glucose Dulbecco’s modified Eagle medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and a penicillin–streptomycin mixture (3%) and cryopreserved in fetal bovine serum containing 10% DMSO. GFP-expressing cells were acquired using a pLKO_047 (Broad Institute) lentiviral vector and puromycin selection. Cell counts were determined with a cell counter (TC20 automated cell counter, (Bio Rad, Hercules, CA, USA).
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