Escobedo-Lucea Carmen, Stojkovic Miodrag
Centro de Investigación Príncipe Felipe, Valencia, Spain.
Methods Mol Biol. 2010;584:55-69. doi: 10.1007/978-1-60761-369-5_3.
The majority of human embryonic stem cell (hESC) lines have been derived and grown using mouse or human feeder cells, or using Matrigel, an animal derivative rich in extracellular matrix (ECM) proteins. However, reliance on feeder layers and animal products limits the manipulation and clinical application of hESC. Alternatively, human fibroblasts produce an ECM which could be employed to coated plates and be easily sterilized. We have shown that hESC grown on this matrix and in the presence of medium conditioned by fibroblast cells maintain markers of pluripotency, including expression of cell surface proteins (SSEA3, SSEA4, TRA-1-60, TRA-1-81), alkaline phosphatase activity, and specific intracellular markers (NANOG, OCT, REX1). Moreover, hESC cultured on this novel human-derived ECM display a normal karyotype. This growth system reduces exposure of hESC to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally facilitating manipulation of hESCs.
大多数人类胚胎干细胞（hESC）系是使用小鼠或人类饲养层细胞，或使用基质胶（一种富含细胞外基质（ECM）蛋白的动物衍生物）来衍生和培养的。然而，对饲养层和动物产品的依赖限制了hESC的操作和临床应用。另外，人成纤维细胞会产生一种细胞外基质，可用于包被培养板且易于灭菌。我们已经表明，在这种基质上生长并在成纤维细胞条件培养基存在的情况下，hESC维持多能性标志物，包括细胞表面蛋白（SSEA3、SSEA4、TRA-1-60、TRA-1-81）的表达、碱性磷酸酶活性以及特定的细胞内标志物（NANOG、OCT、REX1）。此外，在这种新型人类来源的细胞外基质上培养的hESC显示出正常的核型。这种生长系统减少了hESC与饲养层和动物成分的接触，从而降低了病原体污染的风险，并进一步便于对hESC的操作。
