Phenotyping senescent mesenchymal stromal cells using AI image translation

Mesenchymal stromal cells (MSCs) offer promising potential in biomedical research, clinical therapeutics, and immunomodulatory therapies due to their ease of isolation and multipotent, immunoprivileged, and immuno-suppersive properties. Extensive efforts have focused on optimizing the cell isolation and culture methods to generate scalable, therapeutically-relevant MSCs for clinical applications. However, MSC-based therapies are often hindered by cell heterogeneity and inconsistency of therapeutic function caused, in part, by MSC senes-cence. As such, noninvasive and molecular-based MSC characterizations play an essential role in assuring the consistency of MSC functions. Here, we demonstrated that AI image translation algorithms can effectively pre-dict immunofluorescence images of MSC senescence markers from phase contrast images. We showed that the expression level of senescence markers including senescence-associated beta-galactosidase (SABG), p16, p21, and p38 are accurately predicted by deep-learning models for Doxorubicin-induced MSC senescence, irradiation-induced MSC senescence, and replicative MSC senescence. Our AI model distinguished the non-senescent and senescent MSC populations and simultaneously captured the cell-to-cell variability within a pop-ulation. Our microscopy-based phenotyping platform can be integrated with cell culture routines making it an easily accessible tool for MSC engineering and manufacturing.