Coller Hilary A, Khrapko Konstantin, Herrero-Jimenez Pablo, Vatland Janice A, Li-Sucholeiki Xiao-Cheng, Thilly William G
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Mutat Res. 2005 Oct 15;578(1-2):256-71. doi: 10.1016/j.mrfmmm.2005.05.014. Epub 2005 Jul 11.
Tissue maintenance stem cells, as opposed to transition and/or terminal cells in the epithelium, are possible progenitor cells for human tumors, but little is known about their frequency in human tissues. It occurred to us that the colonies of mutants that should be created when a stem cell mutates and transmits the rare mutation to its descendent transition and terminal cells should, given a quantitative mutation assay, define the average number of cells in a maintenance turnover unit and permit calculation of stem cell number. To test this concept we used a combination of high fidelity PCR and constant denaturant capillary electrophoresis to enumerate mitochondrial point mutations and define their number and distribution among multiple small samples of approximately one million cells containing about 400 million copies of mitochondrial DNA. The bulk of the data were best explained by a model in which most stem cells, defined here as long-lived cells, give rise to colonies of approximately 8-128 cells. In addition, we found that about 1.5% of colonies contained hundreds or even thousands of homoplasmic mutant cells. These expanded turnover units suggest the bronchial epithelium may contain large clusters of cells with mutations, and possibly phenotypic alterations as well.
与上皮组织中的过渡细胞和/或终末细胞不同，组织维持干细胞有可能是人类肿瘤的祖细胞，但我们对它们在人体组织中的频率了解甚少。我们想到，当干细胞发生突变并将这种罕见突变传递给其后代过渡细胞和终末细胞时，应该会产生突变体克隆。如果有一个定量突变检测方法，那么这些克隆应该能够确定维持更新单位中的平均细胞数量，并允许计算干细胞数量。为了验证这一概念，我们结合了高保真PCR和恒变性毛细管电泳来计数线粒体点突变，并确定它们在多个约一百万个细胞的小样本中的数量和分布，这些样本包含约4亿个线粒体DNA拷贝。大部分数据最好用一个模型来解释，在这个模型中，大多数在这里被定义为长寿细胞的干细胞会产生大约8 - 128个细胞的克隆。此外，我们发现约1.5%的克隆包含数百甚至数千个同质性突变细胞。这些扩大的更新单位表明支气管上皮可能含有大量带有突变的细胞簇，并且可能也有表型改变。
