Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods
Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods
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DOI：
10.1007/s10616-014-9822-0
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发表时间：
2016-08-01
期刊：
影响因子：
2.2
通讯作者：
Pfeiffer, D.
中科院分区：
文献类型：
--
作者：
Mazza, G.;Rossmanith, E.;Pfeiffer, D.
尽管脐带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并不能促进人脐动脉平滑肌细胞（HUASMC）的分离。为了准确表征HUASMC及其环境中的细胞，在脐带组织切片中分析平滑肌和间充质标志物的表达。然后使用所得标记物谱来评价HUASMC分离和培养方法的质量。HUASMC和血管周-沃顿氏胶质基质细胞（pv-WJSC）显示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CD 90阳性染色。抗CD 10仅pv-WJSC染色。因此，HUASMC可表征为α-SMA+、SM-MHC+、CD 10-细胞，其另外对内皮标志物（CD 31和CD 34）呈阴性。酶促分离提供了纯度为90- 99%的原代HUASMC批次，然而，在标准培养条件下，污染的CD 10+细胞迅速构成总细胞群的80%以上。污染主要是由于HUASMC对细胞培养板的粘附性差，无论不同的蛋白质涂层（纤连蛋白、胶原蛋白I或明胶）如何。HUASMC仅在3D基质中表现出较强的附着力和长期生存能力。外植体分离方法获得的培养物仅具有13- 40%的纯度，具有相当大的CD 10+细胞污染。CD 10+细胞在平滑肌分化培养基中培养后呈梭形形态，α-SMA和SM-MHC表达上调。考虑到HUASMC培养物被CD 10+邻近细胞污染的高风险及其表型相似性，必须进行精确表征，以避免误导性结果。
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 alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as alpha-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 alpha-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.