Characterization of mutational processes in adult stem cells (ASCs) will improve our understanding of aging-related diseases, such as cancer and organ failure, and may ultimately help prevent the development of these diseases. Here, we present a method for cataloging mutations in individual human ASCs without the necessity of using error-prone whole-genome amplification. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for accurate whole-genome sequencing (WGS) analysis. We developed a data-analysis pipeline that identifies with high confidence somatic variants that accumulated in vivo in the original ASC. These genome-wide mutation catalogs are valuable resources for the characterization of the underlying mutational mechanisms. In addition, this protocol can be used to determine the effects of culture conditions or mutagen exposure on mutation accumulation in ASCs in vitro. Here, we describe a protocol for human liver ASCs that can be completed over a period of 3-4 months with hands-on time of ∼5 d.

中文翻译：

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通过类器官培养物的全基因组测序测量单个人类成体干细胞中的突变积累。

成人干细胞 (ASC) 突变过程的特征将提高我们对衰老相关疾病的理解，例如癌症和器官衰竭，并可能最终有助于预防这些疾病的发展。在这里，我们提出了一种对单个人类 ASC 中的突变进行分类的方法，而无需使用容易出错的全基因组扩增。单个 ASC 在体外扩展为克隆类器官培养物，以产生足够的 DNA 用于准确的全基因组测序 (WGS) 分析。我们开发了一个数据分析管道，可以高可信度地识别在原始 ASC 中体内积累的体细胞变异。这些全基因组突变目录是表征潜在突变机制的宝贵资源。此外，该协议可用于确定培养条件或诱变剂暴露对体外 ASCs 突变积累的影响。在这里，我们描述了一个人类肝脏 ASC 的协议，该协议可以在 3-4 个月内完成，动手时间约为 5 天。