(683h) Directing Stem Cell Differentiation By Engineering Cell-Cell Adhesion Pathways

The differentiation fate of a stem cell is controlled by its microenvironment. Although the role of soluble factors (e.g. TGF-β1) in mesenchymal stem cell (MSC) differentiation towards smooth muscle (SMC) lineage has been studied, the role of intracellular adhesion in this process remains elusive. Our previous studies have shown that cell-cell adhesion is necessary to induce MSC differentiation into vascular fate, particularly through the cell surface molecule, OB-cadherin. To further investigate the role of OB-cadherin in MSC proliferation and differentiation potential, we employed a fusion protein of OB-cadherin with the Fc domain (OB-Fc), which allowed us to study cadherin-cadherin interactions in an isolated manner. In particular, OB-Fc is a chimeric homo-dimer consisting of CH2 and CH3 (Constant Heavy-chain) region of FC  and is linked through the region of the antibody to all five extracellular domains of OB cadherin. Initially, IgG-1 which preferentially bound to FC fragment was coated on a hydrophobic plate and afterwards, Bone Marrow Mesenchymal Stem Cells (BM-MSCs) were plated on OB-FC surface. To this end, these cells exhibited increased proliferation (∼5-fold) and suppressed expression of senescence-associated proteins such as p21 over several passages. At the same time, culture on OB-Fc surface enhanced the BM-MSCs differentiation potential towards the SMC lineage as evidenced by the levels of key myogenic markers such as αSMA and CNN1. In particular, engagement of OB-cadherin increased αSMA expression through ROCK pathway and  its downstream effectors such as SRF. On the other hand, knocking down OB-cadherin using shRNA reversed these effects. Finally, tissue constructs prepared from cells that were propagated on OB-Fc exhibited significantly increased vascular contractility and improved mechanical properties. Notably, these results were confirmed in vivo using OB-cadherin knockout mice (OB^-/-). In particular, smooth muscle containing organs from OB^-/- animals e.g. bladder exhibited diminished vascular reactivity, decreased collagen content and significantly decreased mechanical properties. To this end, our results demonstrate that engineering OB-FC containing substrates that mimic the in vivo microenvironment promotes MSC proliferation and differentiation potential into SMC, thereby suggesting that this may be a novel and potentially clinically applicable strategy for directing stem cell fate.