King Charles C, Beattie Gillian M, Lopez Ana D, Hayek Alberto
University of California, Whittier Institute, Department of Pediatrics, San Diego, 9500 Gilman Drive, Center for Molecular Genetics, Room 131, La Jolla, CA 92037-0634, USA.
Regen Med. 2008 Mar;3(2):175-80. doi: 10.2217/17460751.3.2.175.
The purpose of these studies was twofold: to reduce the level of nonhuman, potentially immunogenic sialic acid N-glycolylneuraminic acid (Neu5Gc) in human embryonic stem cells (hESCs) through culture of the cells in the absence of feeder layers; and to determine whether directed differentiation was preserved under these conditions, that is, using exclusively human-derived products.
Using a technique developed in our laboratory to culture hESCs in the absence of feeder layers, all nonhuman cell culture reagents were replaced with recombinant or human-derived reagents. The level of the nonhuman sialic acid (Neu5Gc) was measured by high-performance liquid chromatography and monitored over many passages. Subsequently, the cells were subjected to in vitro differentiation into definitive endoderm by lowering the serum concentrations and elevating the amount of activin A.
Under standard tissue culture conditions using mouse and other animal products, the basal levels of Neu5Gc were measured between 7 and 10%. After the cell culture reagents were changed to all human products, Neu5Gc levels decreased steadily before leveling below 2%. Upon initiation of the differentiation protocol under these cell culture conditions, we observed robust endoderm formation, as measured by fluorescence-activated cell sorting analysis and the appearance of mRNA for markers of definitive endoderm (Sox17, CXCR4, Goosecoid and FoxA2).
Consistent with other findings, elimination of nonhuman products in cell culture of hESCs decreases the levels of nonhuman and potentially immunogenic sialic acid levels. Furthermore, our studies demonstrate that in this feeder layer-free system, hESCs undergo directed differentiation into definitive endoderm.
这些研究有两个目的：通过在无饲养层的条件下培养人胚胎干细胞（hESCs），降低其中非人类的、具有潜在免疫原性的唾液酸N-羟乙酰神经氨酸（Neu5Gc）的水平；并确定在这些条件下（即仅使用人类来源的产品）定向分化是否得以保留。
使用我们实验室开发的在无饲养层条件下培养hESCs的技术，将所有非人类细胞培养试剂替换为重组或人类来源的试剂。通过高效液相色谱法测量非人类唾液酸（Neu5Gc）的水平，并在多个传代过程中进行监测。随后，通过降低血清浓度和增加激活素A的量，使细胞在体外分化为确定内胚层。
在使用小鼠和其他动物产品的标准组织培养条件下，Neu5Gc的基础水平在7%至10%之间。当细胞培养试剂全部更换为人类产品后，Neu5Gc水平稳步下降，最终降至2%以下并趋于平稳。在这些细胞培养条件下启动分化方案后，通过荧光激活细胞分选分析以及确定内胚层标志物（Sox17、CXCR4、Goosecoid和FoxA2）的mRNA出现情况，我们观察到了强大的内胚层形成。
与其他研究结果一致，在hESCs细胞培养中消除非人类产品可降低非人类及潜在免疫原性唾液酸的水平。此外，我们的研究表明，在这个无饲养层系统中，hESCs可定向分化为确定内胚层。
