How to track cellular aging of mesenchymal stromal cells?

Abstract

Mesenchymal stromal cells (MSC) are currently tested in a large number of clinical trials and raise high hope in regenerative medicine. These cells have to be expanded in vitro before transplantation and several studies demonstrated that long-term culture evokes continuous changes in MSC: proliferation rate decays, the cell size increases, differentiation potential is affected, chromosomal instabilities may arise and molecular changes are acquired. Long-term culture of cell preparations might also have therapeutic consequences, although this has hardly been addressed in ongoing trials so far. Reliable therapeutic regimens necessitate quality control of cellular products. This research perspective summarizes available methods to track cellular aging of MSC. We have demonstrated that gene expression changes and epigenetic modifications are continuously acquired during replicative senescence. Molecular analysis of a suitable panel of genes might provide a robust tool to assess efficiency and safety of long-term expansion.

Figure 1.. Continuous gene-expression changes in MSC upon long-term culture.

MSC from human bone marrow were expanded for 11 passages and analyzed by Affymetrix GeneChip technology. Differential gene expression was always determined versus P2. Hierarchical cluster analysis of all expressed genes (19,448 ESTs) revealed continuous changes with higher passages. Hence, molecular changes in replicative senescence do not suddenly occur in late passages, but are acquired in the course of long?term culture.

Figure 2.. Gene expression markers for replicative senescence.

MSC from human bone marrow were either culture expanded as described before in medium-M1 with 2% fetal calf serum (M1, in Heidelberg, Germany [1]; n=3), in culture medium with 10% fetal calf serum (FCS, n=2) or 10% pooled human platelet lysate (pHPL, n=2; both in Graz, Austria [38]), in MEM supplemented with 20% FCS (Innsbruck, Austria [40]; n=2), and in MSCGM (Lonza) culture medium (Rostock, Germany; n=4). Furthermore, MSC from adipose tissue were expanded with 10% pHPL (Aachen, Germany, n=4). RNA was isolated from corresponding early and late passages and analyzed for differential gene expression in PARG1, CDKN2B, MCM3, PTN and p16^ink4a. Primers and methods have been described before [38]. These genes did not facilitate reliable discrimination of senescent cells in all samples but the tendency was consistent in all different MSC preparations.