Induction of smooth muscle from neural crest cells in vitro

Abstract

Vascular smooth muscle cells (VSMC) in the aortic arch arteries are of ectodermal origin, arising mainly from rhombencephalic neural crest (NC); whereas other VSMC are mesodermal in origin, differentiating from local mesenchyme. This lineage has been shown directly in birds, and indirectly for man and other mammals. Though these two precursor cells share the same environment, there are significant differences in the timing and distribution of their respective expressions of the VSMC phenotype. The mechanism of induction of the VSMC phenotype must vary with the lineage of the precursors, but the mechanisms of induction of VSMC from more primitive cells is not known. The differentiation of VSMC from NC cells is the focus of this in vitro investigation. It was hypothesized that the differentiation of NC cells into VSMC is the result of the inductive influence of cytokines, dependent upon a permissive extracellular matrix.

To determine the effect of extracellular matrix molecules upon the differentiation of NC cells into the VSMC phenotype, neural fold explants from stage 9 chicken embryos were grown on substrates of fibronectin, laminin, tenascin, chondroitin-6-sulfate, collagen, and combinations of these; all of these matrix components are found in the migratory pathway and terminus of migration of the NC cells in vivo. VSMC phenotype was defined by the expression of smooth muscle alpha actin (SMAA). SMAA was expressed in all cultures and optimally with fibronectin.

To determine the effect of certain growth factors or cytokines upon NC differentiation, NC fold explants were grown on substrates of fibronectin with only one cytokine per culture medium. Three cytokines were chosen: basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and platelet-derived growth factor (PDGF). Both bFGF and PDGF B-chain were highly effective in promoting growth and differentiation of neural crest-derived VSMC; whereas TGF-beta and PDGF A-chain were ineffective or inhibitory. Although bFGF and PDGF B-chain dimers have been shown to affect VSMC proliferation, this is the first report of their ability to promote VSMC differentiation from precursor cells, and they did so in a dose-dependent manner. The opposite effects of bFGF and TGF-beta in this study are consistent with their known antagonism in other systems. The results of this study strongly support the hypothesis.