(786g) NANOG Restores the Impaired Contractile Function of Senescent Mesenchymal Stem Cells

Contractility is an essential function of tissue engineered constructs containing smooth muscle cells such as blood vessels and bladders. Although, mesenchymal stem cells (MSC) have been extensively used as a source of smooth muscle cells (SMC), recent studies in our laboratory showed deficits in the myogenic differentiation capacity of senescent MSCs due to decrease in the formation of actin filaments. Unfortunately, current strategies for improving the contractile function have been unsuccessful in restoring this function after senescence, since senescent cells failed to respond to extracellular signaling factors. Interestingly recent work in our laboratory showed that ectopic expression of a single pluripotent transcription factor, NANOG, could reverse aging and restore the impaired contractile properties of aged MSC. In this study, we sought to identify the effects of NANOG on the actin filament organization and force generation capacity of MSC in a 3D microenvironment.

To this end, Human Hair Follicle Mesenchymal Stem Cells (hHF-MSCs) were isolated from a 73 year old donor and senesced by serial passaging in vitro. The cells were transduced with a tetracycline regulatable vector that tightly controls the expression of NANOG by the addition of the tetracycline analogue, doxycycline (Dox). The protein and mRNA levels of α-smooth muscle actin (ACTA2) were quantified by western blotting and real time PCR, respectively. Organization of actin filaments were visualized using immunocytochemistry techniques. To measure the biomechanical force and map the biochemical properties of cells in 3D tissues, we utilized micro-fabricated tissue gauges (µTUGs) that contain a pair of flexible micropillars. The statistical significance between two mean values was analyzed using two-tailed unpaired Studentâ??s t-test values of p<0.05 were considered statistically significant.

We found that, cellular senescence impaired the expression of myogenic genes, actin filament organization and the force generation capacity of MSC derived SMC. Notably, expression of NANOG restored all these functions, ultimately reversing cellular senescence. Interestingly, while senescent cells exhibited impaired TGF-β1 pathway, NANOG completely restored this pathway and reversed the loss of contractile force generated by senescent 3D microtissues (Fig. 1). We will show that the mechanism involves activation of SMADs, which were impaired in senescent cells. We also investigated whether NANOG must be continuously present in order to elicit its effects. Despite the short half-life of NANOG protein (2.5±0.5 hr), the effects of NANOG on myogenic gene and protein expression and contractile function were sustained up to 10 days after termination of expression. This result may suggest interesting epigenetic regulation that may provide long-term recovery of the senescent phenotype of 3D tissues. We are currently investigating such epigenetic events.

In conclusion, ectopic expression of the embryonic transcription factor, NANOG, restored actin polymerization and the contractile function of senescent MSC-derived SMC, suggesting that NANOG may be used to enhance the contractile properties of tissue engineered constructs containing SMC, such as bladder and arteries. Given the effects of NANOG on SMC differentiation and contractile fucntion, it is possible that NANOG might also affect the contractile function of other types of muscle such as cardiac or skeletal muscle.