Adipose-Derived Stem Cell Differentiation Triggered by TGFß

Adipose-derived stem cells (ADSCs) are a multipotent mesodermal population of cells that are more abundant than bone marrow-derived mesenchymal stem cells and are easy to harvest from lipoaspirate generated from liposuction of fat cells. Increasing evidence shows that both MSCs and ADSCs have the ability to become vascular cells via differentiation. We are investigating the differentiation of ADSCs into vascular smooth muscle cells (VSMCs). VSMCs play a critical role in blood pressure regulation, angiogenesis, vessel wall maintenance, and influence endothelial cell behavior. The differentiation of ADSCs to VSMCs will be triggered by transforming growth factor beta. TGF-beta signaling activates expression of a large set of VSMC-specific genes that produce smooth-muscle-specific proteins such as alpha smooth muscle actin and calponin. To stimulate VSMC differentiation, ADSCs are treated with differentiation media containing TGFß and cultured for ten days. At days four and ten, treated cells will be analyzed for VSMC differentiation markers and compared to control, non-treated cells. To verify VSMC differentiation, cells will be tested for smooth muscle markers by immunohistochemistry, using VSMC-specific antibodies for alpha smooth muscle actin and calponin. In addition, quantitative RT-PCR will be used to determine gene expression levels using VSMC-specific primers for calponin and alpha smooth muscle actin, which will be normalized to GAPDH. Once we have confirmed that ADSCs have differentiated into VSMCs, we will then examine specific signaling pathways downstream of TGFß that regulate the differentiation process