Restoring the function of Aged Mesenchymal Stem Cells for Vascular Tissue Engineering
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Bone marrow-derived mesenchymal stem cells (BM-MSCs) have high potential as an autologous cell source of vascular progenitors but normal cell function and turnover frequency may decline with age. In this study we set out to study the effects of organismal ageing on the molecular and functional properties of a sub population of BM-MSCs, bone marrow-derived smooth muscle cells (BM-SMCs). To this end, we employed a smooth muscle alpha-actin (αSMA) promoter driving expression of enhanced green fluorescence protein (EGFP) to isolate SMCs from bone marrow of neonatal (nBM-SMCs) or adult (aBM-SMCs) sheep and examined their proliferation potential and contractility. Compared with nBM-SMCs, aBM-SMCs exhibited lower clonogenicity and proliferation potential that could be improved significantly by addition of basic fibroblast growth factor. Vascular constructs from aBM-SMCs showed reduced ability to generate force and contract fibrin hydrogels and this function could be partially restored by addition of transforming growth factor-beta1 (TGF-β1). They also exhibited lower receptor- and non-receptor-mediated vascular contractility and mechanical strength, which was comparable to that of tissue constructs prepared with vascular SMCs from neonatal umbilical veins. In agreement with the contractile properties and mechanical strength of vascular constructs, aBM-SMCs displayed significantly lower expression of αSMA, smoothelin, desmin, type I collagen, and tropoelastin transcripts compared with nBM-SMCs. Collectively, these results show that MSCs originating from older donors suffer from limited proliferative capacity and significantly reduced myogenic differentiation potential. This is a major concern, as the patients most likely to suffer from cardiovascular disease are elderly. Here we tested the hypothesis that a single pluripotency associated transcription factor, namely Nanog, may reverse the proliferation and differentiation potential of BM-MSCs from adult donors. Microarray analysis showed that adult (a)BM-MSCs expressing Nanog clustered close to Nanog-expressing neonatal cells. Nanog markedly upregulated genes involved in cell cycle, DNA replication and DNA damage repair and enhanced the proliferation rate and clonogenic capacity of aBM-MSCs. Notably, Nanog enhanced the myogenic differentiation potential and reversed the effects of organismal aging on the contractile function of aBM-MSCs to a similar level as that of neonatal (n)BM-MSCs. The effect of Nanog on contractility was mediated in part through activation of the TGF-β1 pathway by diffusible factors secreted in the conditioned medium of Nanog-expressing BM-MSCs. At last, we implicated that Nanog also enhanced smooth muscle contractility through JNK activation. Nanog overexpression induced constitutive JNK phosphorylation and blocking JNK with chemical inhibitors or small interfering RNA (siRNA) decreased matrix contraction in BM-MSCs significantly. In addition, we found that JNK activation in Nanog expressing BM-MSCs occurred regardless of TGF-β signaling, indicating those two signaling pathways synergistically contribute to Nanog-mediated contractile function. Overall, our results suggest that Nanog may be used to overcome the effects of organismal aging on aBM-MSCs, thereby increasing the potential of MSCs from aged donors for cellular therapy and tissue regeneration.