Expansion and Characterization of hMSCs

Frozen hMSCs from passage 0 to 2 were delivered from the Tulane Center for Gene Therapy. hMSCs isolated from the bone marrow of multiple de-identified healthy donors from age 19–49 years (Table S1) were collected from plastic adherence, with less than 2% expression of CD3, CD14, CD31, CD45 and CD117, greater than 95% expression of CD73, CD90, CD105 and CD147, while simultaneously possessing tri-lineage differentiation potential upon in vitro induction [36 (link)]. hMSCs (1 × 10⁶ cells/mL/vial) were cryopreserved in a culture media encompassing α-MEM, 2 mM L-glutamine, 30% fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). hMSCs were thawed and seeded at 2500 cells/cm² in a complete culture media containing α-MEM, 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) in a standard culture incubator at 37 °C at 5% CO₂. The culture medium was changed every two days. Cells were grown to 80% confluence and harvested with a 0.25% trypsin/ethylenediaminetetraacetic acid solution (Invitrogen, Grand Island, NY, USA) at 37 °C for 5–7 min. Harvested cells were re-plated at a density of 2500 cells/cm² and sub-cultured up to passage 4–6 for experiments.