Fluorescence-activated cell sorting of senescent fibroblasts

Introduction:

COPD is associated with cellular senescence, hallmarks of which include increased cell size, autofluorescence, senescence associated-β-galactosidase (SA-β-Gal) staining, mitochondrial dysfunction, reduced proliferation and cell cycle inhibitor expression. Dilution of these markers due to proliferation of non-senescent cells makes studying senescence in proliferative cells, such as fibroblasts, challenging.

Aim:

Assess whether senescent fibroblasts can be isolated from a mixed COPD fibroblast population.

Method:

Fibroblasts were isolated from COPD patient lung resections (n=6). Cells were sorted using a FACSAria Fusion into a "senescence-enriched population" (largest and most autofluorescence) and a "non-senescent population" (smallest, least autofluorescence). Senescence was investigated in these populations using SA-β-Gal for senescent cell identification, iCELLigence technology to assess proliferation, Seahorse assay to investigate mitochondrial function and RT-qPCR to measure cell cycle inhibitor expression.

Results:

Senescence-enriched fibroblasts were larger and more autofluorescence and displayed greater SA-β-Gal staining. These cells trended towards reduced proliferation over the first 48h of growth compared to the non-senescent population. Senescence-enriched fibroblasts showed increased basal mitochondrial function (p<0.05), maximal respiration (p<0.05) and ATP production (p=0.06). Senescence-enriched fibroblasts also exhibited elevated p16^INK4a (p<0.05), p21^WAF1/CIP1 (p<0.05) and p15^INK4b (p=0.10) gene expression.

Conclusion:

These data suggest FACS isolate senescent-enriched fibroblasts. This technique can be used to study fibroblast senescence mechanisms and provide therapeutic targets for COPD.