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

Area of Science:

• Cell Biology
• Immunology
• Regenerative Medicine

Background:

• Mesenchymal stromal cells (MSC) are crucial for cell therapy, but their therapeutic efficacy is limited by variable cell quality.
• Prolonged culture of MSC leads to senescence, reducing cell fitness and therapeutic potential.

Purpose of the Study:

• To evaluate the suitability of fluorescence-activated cell sorting (FACS) based on autofluorescence for non-invasively eliminating senescent MSC.
• To assess the impact of autofluorescence-based sorting on MSC characteristics and gene expression.

Main Methods:

• MSC were sorted into low-autofluorescence (LA) and high-autofluorescence (HA) groups using FACS.
• Senescence markers (cell volume, SA-β-Gal assay, gene/protein expression) and differentiation potential were assessed.
• Transcriptional profiles of sorted MSC were analyzed using RNA-Seq.

Main Results:

• LA MSC exhibited reduced cell volume, autofluorescence, and SA-β-Gal activity compared to controls.
• HA MSC showed increased cell volume, autofluorescence, and SA-β-Gal activity.
• No significant differences in replicative senescence or differentiation potential were observed between groups.
• RNA-Seq revealed 68 differentially expressed genes between LA and other groups, with 16 related to senescence, highlighting CXCL12 as a key regulator.

Conclusions:

• Autofluorescence-based MSC sorting is a promising method for enriching functional MSC populations by removing senescent cells.
• This technique holds potential for improving the clinical outcomes of MSC-based therapies by ensuring higher cell quality.