Bone marrow was obtained by flushing the femoral heads and trabecular bone collected from patients undergoing total hip arthroplasty (approved by Institutional Review Board, University of Washington and University of Pittsburgh). After several rounds of rinsing, the hMSCs were plated in T150 flasks (Corning Inc., corning, NY) and cultured in growth medium [GM, Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10 % (v/v) fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA) and 1X antibiotic-antimycotic (anti-anti; Gibco)] supplemented with 1.5 ng/mL fibroblast growth factor-2 (FGF-2; RayBiotech, Norcross, GA). When the cultures reached 70%–80% confluence, the hMSCs were detached with trypsin-0.25% ethylenediaminetetraacetic acid (ThermoFisher, Waltham, MA) and passaged at 1:3 dilution.
The colony formation ability of the hMSCs was evaluated using the colony forming unit (CFU) assay using a standard protocol [31 (link)]. Trilineage (osteogenic, chondrogenic, and adipogenic) cell differentiation was routinely performed in 2D culture to validate the stemness of the pooled hMSCs [32 (link)]. hMSCs isolated from 10 male (average age: 51 years old) and 10 female (average age: 53 years old) donors were pooled before use. All experiments were performed with passage 5 hMSCs.
The colony formation ability of the hMSCs was evaluated using the colony forming unit (CFU) assay using a standard protocol [31 (link)]. Trilineage (osteogenic, chondrogenic, and adipogenic) cell differentiation was routinely performed in 2D culture to validate the stemness of the pooled hMSCs [32 (link)]. hMSCs isolated from 10 male (average age: 51 years old) and 10 female (average age: 53 years old) donors were pooled before use. All experiments were performed with passage 5 hMSCs.