Mazza G, Roßmanith E, Lang-Olip I, Pfeiffer D
Center for Biomedical Technology, Danube University Krems, Dr.-Karl-Dorrek-Str. 30, 3500, Krems, Austria.
Center for Integrated Sensor Systems, Danube University Krems, Dr.-Karl-Dorrek-Str. 30, 3500, Krems, Austria.
Cytotechnology. 2016 Aug;68(4):701-11. doi: 10.1007/s10616-014-9822-0. Epub 2014 Dec 23.
Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton's jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+ , SM-MHC+ , CD10- cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90-99 % purity, yet, under standard culture conditions, contaminant CD10+ cells rapidly constituted more than 80 % of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13-40 % purity with considerable contamination by CD10+ cells. CD10+ cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+ neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results.
尽管脐动脉是血管平滑肌细胞的常见来源，但缺乏可靠的标志物谱不利于人脐动脉平滑肌细胞（HUASMC）的分离。为了准确表征HUASMC及其周围环境中的细胞，对脐带组织切片中平滑肌和间充质标志物的表达进行了分析。然后将所得的标志物谱用于评估HUASMC分离和培养方法的质量。HUASMC和血管周围-华通氏胶基质细胞（pv-WJSC）对α-平滑肌肌动蛋白（α-SMA）、平滑肌肌球蛋白重链（SM-MHC）、结蛋白、波形蛋白和CD90呈阳性染色。抗CD10仅对pv-WJSC染色。因此，HUASMC可被表征为α-SMA+、SM-MHC+、CD10-细胞，这些细胞对内皮标志物（CD31和CD34）也呈阴性。酶法分离得到的原代HUASMC批次纯度为90%-99%，然而，在标准培养条件下，污染的CD10+细胞迅速占总细胞群体的80%以上。污染主要是由于HUASMC对细胞培养板的粘附性差，无论使用何种不同的蛋白质包被（纤连蛋白、I型胶原或明胶）。HUASMC仅在三维基质中表现出强附着和长期活力。外植体分离法获得的培养物纯度仅为13%-40%，且被CD10+细胞大量污染。CD10+细胞呈纺锤样形态，在平滑肌分化培养基中培养时α-SMA和SM-MHC的表达上调。考虑到HUASMC培养物被相邻CD10+细胞污染的风险很高以及它们的表型相似性，必须进行精确表征以避免误导性结果。
