2UCL Institute of Child Health and Great Ormond St. Hospital for Children, 30 Guilford St., London WC1N 1EH, UK.
FASEB J. 2013 Dec;27(12):4853-65. doi: 10.1096/fj.12-218578. Epub 2013 Aug 30.
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-β1. Molecular assays revealed higher level of α smooth muscle actin (α-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 α-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological 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.
功能性平滑肌工程需要分离和扩增平滑肌细胞（SMCs），而对于内脏平滑肌组织，由于尚未明确鉴定出祖细胞，因此这一过程尤其具有挑战性。在此，我们首次表明可以从人羊水干细胞（hAFSCs）中获得有效的 SMCs。从 c-kit(+) hAFSCs 中生成克隆系。使用 PDGF-BB 和 TGF-β1 条件培养基获得向 SM 谱系（SMhAFSCs）的分化。分子分析显示，与 hAFSCs 相比，SMhAFSCs 中 α 平滑肌肌动蛋白（α-SMA）、结蛋白、钙调蛋白和 smoothelin 的水平更高。超微结构分析表明，SMhAFSCs 还在细胞质中呈现出增加的中间丝、致密体和类似于 SMC 的糖原沉积。通过质谱评估 SMhAFSC 代谢显示，与 hAFSCs 相比，葡萄糖氧化更高，对有丝分裂刺激的反应增强。通过慢病毒载体转导 hAFSCs，并在 α-SMA 启动子的控制下表达 ZsGreen 进行膜片钳实验，证明 SMhAFSCs 保留了平滑肌细胞样的电生理特征。最终，SMhAFSCs 的收缩性在单细胞水平和胶原蛋白凝胶上均很明显。总之，我们在这里表明，在选择性培养条件下，hAFSCs 能够产生功能性 SMCs。
