Human amniotic fluid stem cell differentiation along smooth muscle lineage
Human amniotic fluid stem cell differentiation along smooth muscle lineage
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DOI：
10.1096/fj.12-218578
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发表时间：
2013-12-01
期刊：
影响因子：
4.8
通讯作者：
De Coppi, Paolo
中科院分区：
文献类型：
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作者：
Ghionzoli, Marco;Repele, Andrea;De Coppi, Paolo
功能性平滑肌工程需要分离和扩增平滑肌细胞(SMC)，这一过程对内脏平滑肌组织来说尤其具有挑战性，因为其前体细胞尚未明确鉴定。在这里，我们首次证明了从人羊水干细胞(HAFSCs)中可以获得高效的SMC。从c-kit(+)hAFSCs中获得克隆株。在PDGF-BB和转化生长因子-β1的条件下，SMhAFSCs被诱导分化为SM系。分子检测显示SMhAFSCs中α-SMA、结蛋白、钙蛋白和平滑蛋白的表达水平高于hAFSCs。超微结构分析显示，SMhAFSCs也出现在细胞质中，中间丝、致密小体和类似于SMCs的糖原沉积增加。质谱学评价SMhAFSC的代谢情况表明，与hAFSC相比，SMhAFSC的葡萄糖氧化程度更高，对有丝分裂刺激的反应更强。在α-SMA启动子的控制下，将转导的hAFSCs与编码ZsGreen的慢病毒载体进行膜片钳实验，结果显示SMhAFSCs保留了平滑肌细胞样的电生理指纹图谱。最终，SMhAFSCs的收缩能力在单细胞水平和胶原凝胶上都是明显的。综上所述，我们的研究结果表明，在选择性培养条件下，hAFSCs能够分化为具有功能的SMC。
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.