Epigenetic-Senescence-Signature during long-term culture of MSCs. (A) MSCs were isolated form bone marrow (BM) of three patients and sub-cultured during expansion as indicated by the hierarchical trees. (B) Long-term growth curves reveal that cell growth decayed within 30 cumulative population doublings (cPDs). Black triangles indicate subculturing as mentioned above. (C) DNAm analysis at six senescence-associated CpG sites was then performed in various cryopreserved vials by pyrosequencing [22, 24]. Based on these results we calculated predictions for cPDs, days of in vitro culture and passage numbers. These predictions were subsequently compared to real values. (D) Predictions for cPDs, as determined using the Epigenetic-Aging-Signature, were plotted against real culture time to simulate long-term growth curves. Overall, the results correspond to real long-term growth curves even with regard to different subcultures (symbols as depicted in B).