Autofluorescence-based sorting removes senescent cells from mesenchymal stromal cell cultures.

Mesenchymal stromal cells (MSC) are used in cell therapy, but results depend on the unknown quality of cell populations. Extended culture time of MSC increases their senescent levels, leading to a critical loss of cell fitness. Here, we tested the suitability of MSC-sorting based on their FACS autofluorescence profile, for a rapid and non-invasive method of senescent cell elimination. Cells were classified in low- (LA) and high- (HA) autofluorescence groups, and results compared to the original MSC population (control). Three days after sorting, cells were screened by replicative senescence markers (cell volume, SA-β-Gal assay and gene/protein expression) and MSC differentiation assays. The transcriptional profiles of sorted MSC were also analyzed by RNA-Seq. Compared to control, LA cells had 10% lower cell volume and autofluorescence, and 50% less SA-β-Gal + cells. Instead, HA cells had 20% higher cell volume and autofluorescence, and 120% more SA-β-Gal + cells. No changes in replicative senescence and differentiation potentials were observed between all groups. However, 68 genes (16 related to senescence) were significantly differentially expressed (DEG) between LA and other groups. Biological network of DEG identified CXCL12 as topological bottleneck. In summary, MSC sorting may have practical clinical implications to enhance the results of MSC-based therapies.