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The decline in mesenchymal stem cell (MSC) self-renewal and function with aging contributes to diseases associated with impaired osteogenesis. MSC donor age in prolonged culture also limits the therapeutic potential of these cells for tissue engineering and regenerative medicine. Here, we demonstrate an intervention to preserve the immature state MSC and consequently maintain self-renewal and differentiation capacity during in vitro aging. We showed that blocking of phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) prevents the development of an age-related phenotype and maintains MSC morphology of early passage cells with high clonogenic frequency and enhanced proliferative capacity. MSC cultured in the presence of inhibitors of Akt or mTOR also robustly maintain their osteogenic potential, that is otherwise lost during in vitro aging. We further report that these effects may be mediated by induction of expression of pluripotency genes Nanog and Oct-4 and by the reduction in the production of cytoplasmic reactive oxygen species (ROS). Additionally, loss of Akt/mTOR and ROS was accompanied with lower levels of DNA damage. These results provide an insight into mechanisms involved in MSC aging and suggest possible interventions to maintain quiescence and function of MSC prior to in vivo transplantation or as pharmacological agents in diseases associated with loss of MSC function. S tem C ells 2014;32:2256-2266 [ABSTRACT FROM AUTHOR]

Understanding the mechanisms that direct mesenchymal stem cell ( MSC) self-renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet-derived growth factor receptor β ( PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF- BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl-2′-deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by q RT-PCR, and by long-term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/ Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/ Akt and PDGFR-β expression. In addition, PI3K/ Akt downstream signal cascades, m TOR and its associated proteins p70S6K and 4E-BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ-induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self-renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self-renewal strategies. [ABSTRACT FROM AUTHOR]