Mesenchymal stem cells (MSCs) have to be culture expanded to gain relevant cell numbers for therapeutic applications. However, within 2-3 months the proliferation rate of MSCs decays until they ultimately reach a senescent state. This is accompanied by enlarged morphology, reduced expression of surface markers, and decreased differentiation potential. So far it is only scarcely understood how long-term culture affects MSC preparations, and five processes seem to be involved: (1) MSCs are composed of different sub-populations, and due to different proliferation rates the heterogeneity changes in the course of in vitro expansion; (2) cells in culture acquire mutations and other stochastic cellular defects; (3) self-renewal of MSCs may be impaired under culture conditions, leading to gradual differentiation; (4) the number of cell divisions might be restricted (e.g., by loss of telomeres), and (5) replicative senescence might be associated with the aging process of the organism. There is a growing perception that long-term culture has to be taken into account--especially for clinical applications. On the other hand, the state of replicative senescence is poorly defined by the number of population doublings or even by the number of passages. Reliable molecular measures for cellular aging are urgently needed.