Format

Send to

Choose Destination
Nat Protoc. 2018 Jan;13(1):59-78. doi: 10.1038/nprot.2017.111. Epub 2017 Dec 7.

Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures.

Author information

1
Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.
2
Hubrecht Institute for Developmental Biology and Stem Cell Research, KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

Abstract

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.

PMID:
29215633
DOI:
10.1038/nprot.2017.111
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Nature Publishing Group
Loading ...
Support Center