Joshur Ang has completed his Masters of Research (MRes) from University of Manchester and has been working in Agency for Science Technology and Research (A*STAR) Singapore for the past five years.
There is a growing therapeutic interest in human Mesenchymal Stromal Cells (hMSCs), owing to their immunomodulatory properties, differentiation potential and multiple cell sources. To meet clinical demands, transition from planar to scalar technologies is vital for production of industrial grade hMSCs. However, existing methods in detecting cellular senescence are poorly aligned with industrial bioprocesses. Herein we report the discovery of a membrane-permeable fluorescent probe, SenezRedTM, which selectively stains live, senescent hMSCs. Senescent fetal bone marrow hMSC was generated by continuous passaging of hMSC to achieve >120 hours of population doubling time. Cellular senescence was validated using cell cycle analysis and beta-galactosidase assay. Utilizing Diversity-Oriented Fluorescence Library Approach (DOFLA), a library of fluorescent small molecules was screened against proliferative and senescent hMSC. This led to the identification of SenezRedTM, a red fluorescent small molecule (Ex/Em: 631/673 nm) that selectively stains live senescent hMSC in both monolayer and microcarrier cultures. The specificity of SenezRedTM was further validated with beta-galactosidase assay and Ki-67 proliferative staining. Staining conditions were optimized to one hour, significantly reducing the staining time compared to conventional senescent assays. Co-staining of SenezRedTM with Mito-Tracker Green suggested the sub-cellular localisation of the probe in mitochondria. However, further work is required to decipher the detailed mechanism on the selective localisation of SenezRedTM. In summary, we have developed a specific senescent hMSC probe that can potentially be used as a rapid screening tool for quality control purposes of hMSC bioprocessing.