(516c) Ectopic Expression of Nanog Up-Regulates Srf and Reverses the Loss of Myogenic Differentiation Capacity of Human Mesenchymal Stem Cells Due to Senescence

Human Mesenchymal Stem Cells (hMSCs) show great promise as an autologous smooth muscle cell source for vascular tissue engineering and regenerative medicine. However culture senescence and organismal aging reduce the proliferative and myogenic differentiation capacity of MSCs. To address this issue, we recently reported that ectopic expression of Nanog can restore the lost contractile phenotype due to aging and enhance the proliferative potential of ovine MSCs (Han et al., 2012, Stem Cells). In the current study we aim in identifying the molecular mechanism that mediates the rejuvenating effects of Nanog in MSCs. MSCs isolated from hair follicle or bone marrow were transduced with a tetracycline regulatable vector that carries the Nanog gene. This system enables Nanog expression upon Doxycycline (Dox) treatment. After serial passaging, MSCs were induced to senescence and Dox was added to the culture medium. Subsequently, the effect of Nanog on proliferative and myogenic capacity of MSCs were evaluated and compared with early passage (EP) MSCs. Ectopic expression of Nanog in late passage (LP) MSCs significantly increases the proliferation and decreases the percentage of senescent cells as indicated by SA-β-GAL staining. Furthermore, we show that Nanog controls the expression of key regulators of senescence such as cyclin inhibitors and DNA methyltransferases. Next we studied the myogenic differentiation upon Nanog expression. SMC-specific markers such as ACTA2 and Calponin were highly upregulated in Nanog⁺LP MSCs resembling EP MSCs. In agreement, the contractility in response to vasoactive agonists was also enhanced. Interestingly, we also report that Nanog reverses the loss of the expression of key myogenic transcription such as SRF, MRTF-b, SMAD2 and MYOCD due to senescence. Notably, we identified that Nanog co-immunoprecipitatets with SRF and enhances the transcriptionaly activity of the SRF DNA binding site (CArG box). At this framework, SRF knockdown abolished the effect of Nanog on myogenic markers and contractility. On the other hand, over-expression of SRF in LP MSCs enhances the myogenic phenotype resembling the effect of Nanog. Taken together these data suggest that Nanog reverses the effects of senescence on MSC proliferation and myogenic differentiation capacity, thereby increasing the potential of MSC from aged donors for cellular therapy and tissue regeneration.