Proteomic analysis of extracellular matrices used in stem cell culture
Proteomic analysis of extracellular matrices used in stem cell culture
复制标题
DOI：
10.1002/pmic.201100030
复制
发表时间：
2011-10-01
期刊：
影响因子：
3.4
通讯作者：
Lajoie, Gilles A.
中科院分区：
文献类型：
--
作者：
Hughes, Chris S.;Radan, Lida;Lajoie, Gilles A.
已经描述了许多用于人胚胎干细胞（hESC）体外生长的基质。然而，它们的确切组成通常是未知的。关于这些基质的成分的信息将有助于为hESC开发完全确定的生长表面。这些基质通常由以广泛丰度存在的蛋白质混合物组成，使得其表征具有挑战性。在这项研究中，我们进行了蛋白质组学分析的五个以前未表征的基质：CellStart，人基底膜提取物（人BME），StemXVivo，桥人细胞外基质（BridgeECM），和小鼠胚胎成纤维细胞条件基质（MEF-CMTX）。基于使用HeLa细胞裂解物优化的蛋白质组学方案，我们采用强阴离子和阳离子交换色谱和SDS-PAGE相结合的方法对五种复杂的细胞外基质（ECM）样品进行了分析。对于这些基质中的每一种，我们识别出许多蛋白质，表明它们的复杂性。我们还将这些结果与生长基质Matrigel（TM）的类似蛋白质组学分析进行了比较。从这些分析中，我们观察到纤连蛋白是几乎所有hESC支持基质的主要成分。这一观察结果导致研究纤连蛋白作为hESC生长的限定ECM的适用性。我们发现，纤连蛋白促进维持多能H9和CA 1人胚胎干细胞在未分化状态使用mTeSR 1培养基。这一发现验证了用于hESC生长的表征基质在揭示在普遍适用的限定系统中培养hESC所需的ECM组分中的效用。
Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However, their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study, we performed the proteomic analysis of five previously uncharacterized matrices: CellStart, Human Basement Membrane Extract (Human BME), StemXVivo, Bridge Human Extracellular Matrix (BridgeECM), and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells, we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices, we identify numerous proteins, indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix, Matrigel (TM). From these analyses, we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system.