| Smooth muscle formation and function is critical in development and postnatal life. Hence studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function providing a potential cell source for tissue engineering and a model to explore the molecular regulation of SMC specification. TGF-beta1 alone or combined with PDGF-BB in serum free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Day 6 MAPC-SMCs demonstrate functional L-type calcium channels and generate force in response to known SMC agonists. These results provide evidence that MAPC-SMC are highly similar to neonatal SMCs, and therefore we initiated studies to define the molecular mechanisms underlying SMC differentiation, by evaluating the expressed gene profile of MAPCs from mouse and rat that were induced to differentiate to SMCs with TGF-beta1. 457 genes were found differentially expressed in both species, of which more than 50 were transcription factors (TFs). To determine the role of the transcription factors in SMC differentiation, a rapid throughput system was needed and we chose gene knockdown and overexpression in the Danio rerio (zebrafish) model. Until now, presence of vascular smooth muscle in zebrafish has been controversial. However, here we provide evidence using in-situ hybridization for several smooth muscle specific genes, that vascular and visceral smooth muscle has developed by day 3 of zebrafish development, which should allow for high throughput screening for the role of transcription factors and other differentially expressed genes from the MAPC-SMC array analysis. Overall, this thesis has developed two models of SMC differentiation that should now provide the opportunity to further define the mechanisms underlying SMC specification, vascular and visceral SMC specification and differentiation, the contribution of SMCs to vasculogenesis in vivo, and the abnormal function of SMCs in disease states, aside from renewable populations of functional SMCs that can be used in tissue engineering applications as well as in vivo therapies. |
