Expansion and Characterization of Human Bone Marrow-Derived MSCs

A total of 8 human bone marrow-derived MSCs from 8 donors were purchased from ALLCELLS and Lonza at passage 1 (Additional file 1: Table S1). By flow cytometry, all MSCs revealed positive expression for CD29, CD44, CD73, CD90, CD105, and CD166 [31 (link), 38 (link)]. MSCs were expanded in medium containing alpha-MEM (Invitrogen), 10% FBS (Atlanta Biologics), 1% L-glutamine (Invitrogen), and 1% penicillin G and streptomycin sulfate (Invitrogen) under a humidified atmosphere of 5% CO₂ at 37 °C as previously reported [31 (link)]. Upon reaching 80% confluency, MSCs were detached from the flask using 0.25% trypsin/1 mM EDTA solution (Invitrogen) and replated at 60 cells/cm². MSCs were expanded to passage 7. Only two donor’s MSCs, 127756 and PCBM1655 did not reach passage 7. The second set of MSCs (n = 7) and mesoderm cells lines (n = 4) were expanded from passage 4 to 8 in marrow stromal cell growth medium from Cell Applications (San Diego, CA) as described in Bellayr et al. (2016) [37 (link)]. Cancer cell lines were purchased from ATCC (Manassas, VA) and cultured according to the manufacturer’s instructions.