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

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.

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Figure 1: Schematic overview of the experimental setup.
Figure 2: Schematic diagram of the protocol.
Figure 3: Theoretical variant allele frequency (VAF) of mutations in a clonal organoid culture.
Figure 4: FACS plots depicting gating strategies for sorting single cells from a human liver organoid culture in Step 12.
Figure 5: Plating cells at a limited dilution.
Figure 6: Variant allele frequency (VAF) density plots for 4 organoid cultures.
Figure 7: SNV accumulation measurements of four human liver ASCs of two donors aged 46 (Donor 1) and 30 years (Donor 2) for Routes A and B.

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Acknowledgements

The authors thank J. de Ligt for his input on the CNV analysis, and J.F. van Velzen for his input on the FACS procedures. This study was financially supported by a Zenith grant from the Netherlands Genomics Initiative (935.12.003) and funding from the NWO Zwaartekracht program Cancer Genomics.nl to E.C., and funding from Worldwide Cancer Research (WCR, grant no. 16-0193) to R.v.B.

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Authors and Affiliations

Authors

Contributions

M.J., F.B., H.C., R.v.B. and E.C. wrote the manuscript. M.J., R.v.B. and V.S. developed the wet lab protocol, and N.B. tested the protocol. The bioinformatics pipeline was developed by F.B. and R.v.B., implemented by F.B. and tested by S.B. and R.J.

Corresponding authors

Correspondence to Ruben van Boxtel or Edwin Cuppen.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Outgrowth potential for organoid formation after picking clonal organoids.

Each dot represents a single human donor. There is no correlation between the age of the human donor and the number of picked organoids that were expanded (correlation = -0.041, p-value = 0.482).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Table 1. (PDF 307 kb)

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Jager, M., Blokzijl, F., Sasselli, V. et al. Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures. Nat Protoc 13, 59–78 (2018). https://doi.org/10.1038/nprot.2017.111

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