(229d) NANOG Rejuvenates the Impaired Extracellular Matrix Expression in Senescent Cells and Thus Restores the Decreased Mechanical Properties of Engineered Tissues
To study the effect of NANOG on aged cells, we applied two widely established models of aging i.e. replicative senescence of human Mesenchymal Stem Cells (MSC), and myofibroblasts derived from Hutchinsonâ??s Guilford Progeria Syndrome (HGPS) patients. Human Mesenchymal Stem Cells (MSC) were induced to senescence by serially passaging. To express NANOG, cells were transduced with a tetracycline-inducible virus, which allowed expression of NANOG only in the presence of Doxycycline (Dox). Upon senescence, cells were treated with DOX (LPN) and the production of ECM was evaluated and compared to Early passage (EP) and senescent cells (Late Passage or LP). Similar experiments were also performed with HGPS derived cells.
Our results show that senescence reduced the production of both Collagen 3 and Elastin. In agreement, tissues engineered using senescent cells showed inferior mechanical properties. Interestingly, NANOG restored the capacity of senescent cells to express ECM molecules and increased the mechanical properties of engineered tissues. Next we performed RNA-seq to identify the mechanism mediating the effects of NANOG on ECM production. We found that NANOG altered the expression of genes that are part of the TGFbeta pathway, which is known to be necessary in ECM production. In agreement, we discovered that NANOG restored the activity of the TGFbeta pathway that was impaired in senescent cells, whereas inhibition of the TGFbeta pathway with either shRNA (SMAD2, SMAD3, SMAD4) or chemical inhibitor (SB431542) abolished the effects of NANOG. Conversely, overexpression of SMAD2 and SMAD3 increased ECM production.
In conclusion, our study provides a novel strategy to restore the impaired production of ECM, which may have broad applications in tissue regeneration and anti-aging treatments.