Mesenchymal stromal cell (MSC) therapy represents a growing therapeutic and commercial value proposition, owing to their paracrine effects, immmunomodulatory properties, and regenerative potential. Developing bioassays of MSC potency that inform biological traits relevant for therapeutic strategy are critical. Further, a shift from planar (culture flasks) to scalar technologies (controlled bioreactors) is increasingly appreciated to ensure functional consistency between product lots, in addition to better process control. Yet, existing methods for stem cell quality-control are resource-intensive and suffer poor alignment with scalar bioprocesses, as they fail to provide for a quick, real time, in-process potency measurement. To this end, we have developed a fluorescent probe for detecting potent/proliferative MSCs, and yet another probe for detecting senescent MSCs, which allow for in-process analysis of stem cell potency and senescence. High-throughput screening of in-house, cell-permeable, auto-fluorescent compound library was performed using proliferative, potent human MSC population, and functionally inferior, senescent MSCs. Potency of MSC populations was qualified by studying their characteristic surface marker profiles, tri-lineage differentiation potential, secretome analysis and tubulation assays. MSC senescence was qualified by assessing growth kinetics, cell-cycle & CFU-F analysis, β-galactosidase staining, and senescence associated secretory phenotype. Through such screening, we have established live, fluorescent probes for specific detection of both proliferative, potent MSCs, and senescent, less-potent human MSCs. These probes have also been validated using human MSCs from various sources such as bone-marrow, adipose tissue and umbilical cord. Merits of our strategy are in a specific, reproducible, potency measure that informs product attributes, following ISCT's minimal criteria, and which can be extended to include adequate controls for assay validation. Hence these probes could help inform bioprocess optimization strategies for developing potent stem cell products, and potentially aid in the identification of high-quality stem-cell sources, and/or donors.
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