Sinha Sanjay, Wamhoff Brian R, Hoofnagle Mark H, Thomas James, Neppl Ronald L, Deering Thomas, Helmke Brian P, Bowles Douglas K, Somlyo Avril V, Owens Gary K
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, 22908-0736, USA.
Stem Cells. 2006 Jul;24(7):1678-88. doi: 10.1634/stemcells.2006-0002. Epub 2006 Apr 6.
The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.
本研究的目的是开发一种从胚胎干细胞（ESC）中获得纯化收缩性平滑肌细胞（SMC）群体的方法，并对其功能进行表征。构建了转基因ESC系，其在平滑肌α-肌动蛋白（SMαα）或平滑肌肌球蛋白重链（SM-MHC）启动子的控制下稳定表达嘌呤霉素抗性基因。然后通过过夜或3天的阴性选择从胚状体中纯化SMC。纯化的SMC通过免疫荧光、免疫印迹、定量逆转录-聚合酶链反应和流式细胞术表达多种SMC标志物，分别命名为APSCs（SMαα-嘌呤霉素选择的细胞）或MPSCs（SM-MHC-嘌呤霉素选择的细胞）。两种SMC系均表现出激动剂诱导的Ca（2+）瞬变，表达功能性Ca（2+）通道，并在胶原凝胶中聚集并用血管活性激动剂（如内皮素-1）刺激或对KCl去极化作出反应时产生收缩力。重要的是，皮下注射经过18小时嘌呤霉素选择的APSCs或MPSCs会导致畸胎瘤的形成，这可能是由于多能干细胞的残留污染所致。相比之下，经过3天嘌呤霉素长时间选择的APSCs或MPSCs在体内未形成畸胎瘤。这些研究首次描述了一种从ESC中生成相对纯化的SMC群体的方法，这些SMC对血管活性激动剂表现出适当的兴奋和收缩反应。然而，研究还表明，即使经过长时间分化，ESC衍生的细胞系仍有发生畸胎瘤的可能性，这突出表明在未来使用ESC衍生物（无论是SMC还是其他细胞类型）进行组织工程应用的研究中，迫切需要有效的方法将分化细胞与残留的多能前体分离。
