(104a) NANOG Restores Collagen Type III Production in Aged Stem Cells
To study the effect of NANOG on aged cells, we employed two widely established models of aging i.e. replicative senescence of human Mesenchymal Stem Cells (MSC), and myofibroblasts derived from patients suffering from Hutchinsonâs Guilford Progeria Syndrome (HGPS), a premature aging syndrome. MSC were induced to senescence by serially passaging. To express NANOG, cells were transduced with a tetracycline-regulatable lentivirus, which enabled expression of NANOG by addition of Doxycycline (Dox) after cells reached senescence. Production of ECM was evaluated in NANOG expressing senescent cells and young and control culture senescent and HGPS cells.
Our results showed that the level of Col3 diminished in senescent cells but was completely restored by ectopic expression of NANOG. To further explore the underlying molecular mechanism, we performed RNA sequencing and Gene Ontology and Ingenuity Pathway Analysis, which showed that the TGF-Î²signaling pathway was activated by NANOG. Genes associated with the activation of TGF-Î² pathway such as SMAD2/3, TGFb2, TGFbR1 and LTBP1 were upregulated, while negative regulators of the pathway Smad6, Smad7 and Smurf2 were downregulated upon NANOG expression. These results were verified with experiments that employed chemical inhibitors and shRNA knockdown. NANOG increased the expression and nuclear accumulation of Smad2/3 leading to increased Col3 transcription. ChiP-Seq experiments revealed that NANOG also bound to the SMAD2 and SMAD3 promoters, which was also verified by immunoprecipitation with antibodies against Smad2/3. In addition, loss and gain of function experiments showed that SMAD3 was mostly responsible for mediating the effects of NANOG in increasing Col3 synthesis.
In conclusion, we report that NANOG restored production of Col3, which was impaired by cellular aging, and suggests novel strategies to restore the impaired ECM production and biomechanical function of aged tissues including skin and arteries, with broad applications in tissue regeneration and potentially anti-aging treatments.