Autofluorescence and cell size-based sorting eliminates senescent cells in synovial mesenchymal stem cells from patients with osteoarthritis.

The presence of senescent cells in synovial mesenchymal stem cell (MSC) preparations reduces the therapeutic efficacy of MSCs as a treatment for osteoarthritis (OA). Senolytic drugs can selectively kill senescent cells; however, their safety profiles remain a major concern. This study investigated whether cell sorting based on the increased autofluorescence (AF) and cell size of senescent MSCs could selectively eliminate senescent cells from human synovial MSC preparations derived from OA patients. Synovial MSCs isolated from six OA patients undergoing total knee arthroplasty were divided into three fractions using a fluorescence-activated cell sorter according to their AF and forward scatter (FSC) intensity: AFhigh, AFlowFSChigh, and AFlowFSClow. The cell diameter, morphology, and senescence-associated β-galactosidase (SA-β-gal) activity were evaluated as indicators of cellular senescence, and colony-forming ability and trilineage differentiation potentials (adipogenesis, osteogenesis, and chondrogenesis) were assessed as indicators of stem cell function. The AFlowFSClow fraction had a smaller cell diameter, less enlarged and flattened morphology, and lower SA-β-gal positivity rate when compared with the AFhigh and AFlowFSChigh fractions. The AFlowFSClow cells formed more colonies and exhibited enhanced chondrogenic differentiation ability, as indicated by stronger safranin O staining, higher glycosaminoglycan/DNA ratios, and upregulation of chondrogenic genes. Adipogenic and osteogenic potentials were comparable among the three fractions. Sorting human synovial MSCs based on AF and cell size effectively eliminated senescent cells and enhanced the stem cell function of the MSC preparation. This drug-free strategy will improve both the safety and therapeutic efficacy of synovial MSC-based treatments for OA.