Ageing analysis of mesenchymal stem cells: improvement of stem cell therapies

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair owing to their properties. For most therapeutic applications, expansion of MSCs in vitro is often required in order to produce adequate numbers of cells. Moreover, cells from elderly donors are used in autologous stem cell therapies, for the treatment of many diseases. It is now known that cellular (prolonged expansion) and chronological (donor) ageing of MSCs have a negative impact on MSCs properties, causing continuous alterations in these cells. This work will focus on understanding the ageing process of MSCs. For this purpose, dental pulp stem cells (DPSCs) were isolated from 13 individual donors of various ages and expanded in cultures, until senescence occured. Firstly, the growth kinetics of these cells were studied to understand the impact of chronological and cellular ageing upon DPSCs expansion. My results suggest that the cells ceased proliferation after about 4 months in culture. It is also worth pointing out that pronounced variation in growth kinetics between the donors was observed. It was demonstrated that DPSCs derived from younger donors had better proliferation rate, generating more cells than those from older donors. Based on the results in this study, a cut off point of 23yrs old was set as ageing for DPSCs. This was followed by identification of new biomarkers or combinations of existing ones that accurately predict the ageing state of MSCs and their therapeutic potential. Study of changes in the volatile profile of hMSCs upon senescence were described for the first time. In addition, alterations in other known biomarkers of senescence were analysed and panels with the most prominent of these biomarkers were created. These panels provide a robust and high-throughput approach to precisely identify and quantify senescent cells in culture, thereby allowing the setting of the expansion limits for effective and safe MSCs therapies. Finally, the effect of various senolytic drugs on DPSCs was tested in this study. These drugs can selectively kill senescent cells. Out of the seven different drugs tested, two demonstrated a senolytic effect with an effective reduction of some of the senescent phenotypes in DPSCs population.