Human amniotic fluid stem cell differentiation along smooth muscle lineage

Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-beta 1. Molecular assays revealed higher level of alpha smooth muscle actin (alpha-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the alpha-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electro-physiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.