Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin alpha1/beta1/alphav and PDGF receptor pathways.

Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1(+)) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1(+) progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6-90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1(+) progenitors with antibodies against integrin alpha(1), alpha(v), and beta(1), but not beta(3), inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor-beta significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (alpha(1), beta(1), and alpha(v))-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor-beta signaling pathways are involved in SMC differentiation.