Directing Mesenchymal Stem Cell Fate toward Smooth Muscle Lineage
Alimperti, Styliani M.
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Mesenchymal stem cells have tremendous therapeutic potential in regenerative medicine and show excellent safety record in clinical applications. MSCs can differentiate into osteogenic, adipogenic, chondrogenic, and myogenic lineage under appropriate conditions, and therefore have been used as a therapeutic tool to treat disorders. The induction of mesenchymal stem cells (MSCs) toward smooth muscle lineage is a complex system since many genes are involved during this process. Loss of gene function is a valuable tool for screening genes involved in Mesenchymal Stem Cell (MSC) differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis such as immunostaining and therefore, quantitative, real-time measurement of the process is difficult. To overcome these limitations, we engineered a shRNA encoding Lentiviral Dual Promoter Vector (sh-LVDP) that allows real-time monitoring of MSC differentiation and simultaneous gene knockdown thereby, providing quantitative assessment of the effect of various pathways in this process. In this vector, the activity of the αSMA promoter was measured by Green Fluorescent Intensity (GFI) and was used as an indicator of myogenic differentiation. The constitutive expression of DsRed was used to measure transduction efficiency and normalize the αSMA promoter activity. Finally, different shRNAs were encoded by a doxycycline regulatable H1 promoter within the viral 3' Long Terminal Repeat (LTR). Our data indicates that real time monitoring of gene expression profiles using sh-LVDP with concomitant gene knockdown provides a useful tool for deciphering gene regulatory networks of complex biological processes such as stem cell differentiation. Specifically, using the sh-LVDP, we identified the role of cadherins through the adherens junctions (AJs) formation during smooth muscle differentiation. The microenvironment of a stem cell often dictates its differentiation fate. 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 inter-cellular adhesion in this process remains elusive. To this end, we examined how cell-cell communication controls the fate of MSC towards smooth muscle cells (SMC). Specifically, we identified the molecular mechanism of MSC differentiation into vascular fate during cell-cell contact through a molecule called Cadherin-11. We further applied this study in-vivo and we confirmed our hypothesis in a mice model. Initially, we observed that increased cell density increased adherens junction (AJ) formation and at the same time, high cell density increased expression of myogenic markers such as αSMA as well as N-cadherin and Cadherin-11. Interestingly, we found that Cadherin-11 mediated SMC differentiation through activation of serum response factor (SRF) via ROCK pathway. Also, myogenic differentiation proceeded even in the absence of TGF-β receptor, while exogenous TGF-β1 failed to promote SMC differentiation in the absence of Cadherin-11. Engagement of Cadherin-11 increased its own expression through SRF, suggesting the presence of an auto regulatory feedback loop that regulated SMC differentiation. Notably, SMC-containing tissues such as bladder and arteries from Cdh11-/- mice showed significantly reduced levels of SMC proteins and exhibited diminished contractility and mechanical properties. To conclude, we revealed a previously unknown role of Cadherin-11 based AJs in the process of SMC regeneration and shed light into the mechanism of MSC differentiation with potential implications for the development of strategies to understand and control stem cell fate decisions. Although cell-cell contact is necessary to induce MSC differentiation into vascular fate, particularly through the cell surface molecule, Cadherin-11, the effect of Cadherin-11 engagement has not single out. To investigate the role of Cadherin-11 in MSC differentiation potential, we designed a surface with exposed Cadherin-11 domains, which allowed us to study cadherin-cadherin interactions in an isolated manner. MSCs cultured on Cad-11-FC surface enhanced the differentiation potential of MSCs towards the SMC lineage as evidenced by the levels of key myogenic markers such as αSMA and CNN1. Also, the differentiated MSCs cultured on Cad-11-FC surface exhibited high levels of collagen indicating that Cadherin-11 may play important role in rigidity and elasticity of the SMC containing organs. Tissue constructs prepared from cells lacking Cadherin-11 exhibited lower vascular contractility and mechanical properties. Notably, these results were confirmed in vivo using Cadherin-11 knock-out mice (Cdh11-/-). In particular, smooth muscle containing organs from Cadh11-/- animals e.g. blood vessels and bladder exhibited diminished vascular reactivity, decreased collagen content and significantly decreased mechanical properties. To further investigate, the mechanism of collagen regulation by Cadherin-11, we found that Cadherin-11 mediated collagen secretion and mechanical properties through ROCK1/2 pathway.