Exploring Cellular Fitness Gradients In Adipose-derived Mesenchymal Stem Cells: An Analysis Of Individual And Pooled Settings.

Exploring Cellular Fitness Gradients in Adipose-Derived Mesenchymal Stem Cells: An Analysis of Individual and Pooled Settings Adipose-derived mesenchymal stem cells (MSCs) are increasingly recognized for their promising potential in regenerative medicine. Yet, the influence of individual cellular fitness on their functional outcomes remains a crucial area of investigation. This study systematically evaluates the impact of cellular fitness on adipose-derived MSCs, employing a range of assays and categorizing cells into distinct cellular fitness groups. In the initial phase of the study, MSCs collected from nine human donors underwent the MTS assay to assess cell viability, the colony-forming unit (CFU) assay to measure clonogenic potential, and the population doubling time (PDT) calculation to evaluate proliferative capacity. Based on the results, cells were categorized into three cellular fitness groups: low, middle, and high. Building upon these classifications, the second phase involved the creation of cell pools. Three cell pools were formed, each comprising three donors from their respective low, middle, and high-fitness groups. This approach allowed for the examination of collective effects within each pool. Subsequently, experiments were carried out simultaneously for both individual donors and cell pools, by conducting an in-depth characterization that included cell function studies such as cell migration studies, cell senescence measurement, osteogenic differentiation potential of the cells, as well as the MTS assay and PDT calculation. Statistical analyses, particularly one-way ANOVA, uncovered interesting findings. As expected, in the MTS assay, the high-fitness group demonstrated statistically significant increased cell viability compared to the low and middle groups. Accordingly, this group exhibited significantly shorter PDT compared to both other groups. Furthermore, the cell senescence markers showed statistically significant lower values in the high-fitness group compared to both other groups. However, osteogenic differentiation and cell migration studies through scratch assay did not show statistical significance across groups. Importantly, the results of the pooled cells corresponded well to the results of the individual donor cells within fitness groups. In conclusion, this comprehensive analysis provides valuable insights into the influence of cellular fitness on adipose-derived MSCs. The observed lower senescence rate in the high-fitness group suggests a favorable condition where cells within this group exhibit an extended duration before entering senescence. These results are indicative of potential superiority in cell health and functionality. Moreover, the cell pools were indeed representative for the individual donors within their respective cellular fitness group. However, this may change when pooling cells from different fitness groups. Current investigations therefore include utilizing cell pools with diverse donor fitness levels, to explore how donor composition influences results, aiming to assess if the potency values would then still align with individual donor means. This study lays the groundwork for further exploration of the intricate interplay between cellular fitness and the therapeutic potential of adipose-derived MSCs.