PMC:1523200 / 14144-17384 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1523200","sourcedb":"PMC","sourceid":"1523200","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1523200","text":"Human feeders and hESCs can be readily distinguished and contamination can be readily assessed\nFor all samples, we conducted an unsupervised one-way hierarchical clustering analysis. The clustering analysis was based on the average linkage and Euclidean distances as the similarity metric using differentially expressed genes identified by ANOVA (P \u003c 0.05). The analysis revealed the underlying features and variation patterns of gene expression in each cell types. Figure 2 shows results of the cluster analysis of relative gene expression in selected samples. As one of our purposes of this study was to distinguish between human fibroblast feeders cells and hESCs and hEBs, wishing to readily detect feeder contamination in hESCs, we included one of the human feeder cells HS27 (ATCC) in this study. We have been using HS27 as feeder cells for H9 hESCs for more than two years and all hESCs grown on HS27 had normal karyotype, expressed all undifferentiated markers, and made teratomas with all germ layers (data not show). The global pairwise comparison clearly showed that human feeders were far more dissimilar to hESCs than hESCs grown in different laboratories, hESCs compared to their differentiated EBs that contained mesodermal tissue, and hESCs compared to the karyotypically variant hESC line BG01V. Pairwise comparisons of human feeders with hESCs resulted in a correlation coefficient of 0.66, which was less than the correlation coefficient of 0.71–0.74 observed between hESCs and their corresponding EBs. The large difference between human feeders and hESCs suggested that it would be possible to identify markers that were robust and reliable in distinguishing the two populations, and these markers would be sufficiently sensitive in detecting contamination of feeders. We examined the data to develop a list of genes that had high levels of expression in human feeder cells maintained in hESC medium but whose expression was low or absent in either ESCs or EBs. The absence of expression in EBs was used as a control for spontaneous differentiation of ESC colonies (including mesodermal differentiation) which may occur and the markers selected should be able to distinguish between these two events. A complete list of genes expressed at least ten-fold higher in human feeders is provided in Figure 3. Quantitative RT-PCR (qPCR) was used to verify the fold change of the expression of 4 genes, including THBS1, MMP3, TNFRSF11B and KRTHA4 (Figure 3C). Further confirmation can also be done using immunocytochemistry, as antibodies against these genes are commercially available.\nFigure 3 Human fibroblast feeder cells can be distinguished from hESCs and EBs. Bead array identified lists of genes that were uniquely expressed in human fibroblast feeders as compared to hESCs (A) and hEBs (B). The four genes whose expression was confirmed by qPCR (C) were in bold. In the graph (C), gene expression of each gene in feeder cells was designated as 1 fold and the bars represented fold decrease for each gene. Thus this comparison allowed us to distinguish between hESCs and human feeders and identify candidate markers that could detect feeder cell contamination should human feeders be used in the propagation of hESCs.","divisions":[{"label":"Title","span":{"begin":0,"end":94}},{"label":"Figure 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