Abstract
Background aims. Mesenchymal stromal cells (MSC) are heterogeneous and only a subset possesses multipotent differentiation potential. It has been proven that long-term culture has functional implications for MSC. However, little is known how the composition of subpopulation changes during culture expansion. Methods. We addressed the heterogeneity of MSC using limiting-dilution assays at subsequent passages. In addition, we used a cellular automaton model to simulate population dynamics under the assumption of mixed numbers of remaining cell divisions until replicative senescence. The composition of cells with adipogenic or osteogenic differentiation potential during expansion was also determined at subsequent passages. Results. Not every cell was capable of colony formation upon passaging. Notably, the number of fibroblastoid colony-forming units (CFU-f) decreased continuously, with a rapid decay within early passages. Therefore the CFU-f frequency might be used as an indicator of the population doublings remaining before entering the senescent state. Predictions of the cellular automaton model suited the experimental data best if most cells were already close to their replicative limit by the time of culture initiation. Analysis of differentiated clones revealed that subsets with very high levels of adipogenic or osteogenic differentiation capacity were only observed at early passages. Conclusions. These data support the notion of heterogeneity in MSC, and also with regard to replicative senescence. The composition of subpopulations changes during culture expansion and clonogenic subsets, especially those with the highest differentiation capacity, decrease already at early passages.
Acknowledgments
We are grateful to Anna Marciniak-Czochra for valuable input for the cellular automaton model. This work was supported by the excellence initiative of the German federal and state governments within the START-Program of the Faculty of Medicine, RWTH Aachen (WW), by the Else Kröner Fresenius Foundation (WW), the Academy of Sciences and Humanities, Heidelberg (WIN-Kolleg; PH), by the German Research Foundation (DFG) within the program 873 (AH, TS), and by the state North Rhine Westphalia within the BioNRW2 project ‘StemCellFactory’ (WW) and by the Stem Cell Network NRW (WW).
Declaration of interest: The authors have no conflict of interest.