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.

5-Fluorouracil treatment induces characteristic T>G mutations in human cancer

“…Isogenic healthy human small intestinal organoids were cultured as described previously (Jager et al 2018). In short, organoids were grown on Complete Human Intestinal Organoid (CHIO) medium, supplemented with 30% Adv+++ (Advanced DMEM F12 [Thermofisher], supplemented with glutamax [1%, Thermofisher], hepes [10 mM, Thermofisher], penicillin/streptomycin [1%, Thermofisher]), in house produced Wnt (50%) (Broutier et al 2016) and R-spondin (20%) (Broutier et al 2016), B27 supplement (1x, Thermofisher), nicotinamide (10 mM Sigma), Nacetylcysteine (1.25 mM, Sigma), Primocin (0.1 mg/ml, Invivogen), A83-01 (0.5 μM, Tocris Bioscience), recombinant noggin (0.1 μg/ml, Peprotech), SB202190 (10 μM, Sigma) and hEGF (50 ng/ml, Peprotech).…”

“…Unlike these anticancer treatments, and in spite of its mutagenic potential, 5-FU could thus far not be linked to any mutational signature using these systematic cancer cohort analyses.Here we assessed the mutational consequences of fluoropyrimidines by exposing organoids of healthy intestinal stem cells to 5-FU followed by genome-wide analysis of single cells. For this, we used a previously described highly sensitive approach based on clonal expansion of individual cells followed by whole genome sequencing for mutational spectrum analysis (Jager et al 2018;Blokzijl et al 2016). In vitro findings were subsequently validated by exploration of mutational patterns in breast and colorectal cancer patients who have had previous fluoropyrimidine treatments.…”

Early divergence of mutational mechanisms drives genetic heterogeneity of fetal tissues

“…Measuring extremely low mutation loads in individual cells is challenging, due to the high noise 10 rates that are associated with single-cell DNA sequencing techniques that typically involve errorprone amplification methods (9). To circumvent this hurdle, we studied mutation accumulation in individual SC cells, by expanding them in vitro as organoids before whole genome sequencing and bioinformatic filtering for clonal variants -a method which we previously applied successfully to identify somatic variants in adult SCs from different tissues and donors of 15 different ages (6,10). Using routine organoid culture conditions (11,12), we successfully derived SC lines from the fetal liver and intestine of four fetuses at weeks 15, 17, and 22 (n=2) of gestation.…”

“…None of these genes are involved in cell proliferation or have been causally implicated in cancer according to the COSMIC cancer gene census (14), suggesting that there was no positive selection for cells with functional somatic mutations that confer a selective advantage. 10…”

Exploration of Genetic Variations through Single‐cell Whole‐genome Sequencing in the Model Ciliate Tetrahymena thermophila

“…So far, single-cell WGA and sequencing have led to important progress in understanding ciliate genome biology and evolution, including the discovery of diverse genomic features such as chromosome copy number variation and gene scrambling in some nonmodel ciliate taxa (Maurer-Alcal a et al 2018) and resolution of a longstanding debate on ciliate phylogeny (Gentekaki et al 2017). However, to our knowledge, no study has yet detected genetic variants in ciliates at the single-cell level through WGA and sequencing, although these techniques are well established in human cell lines (Hou et al 2015;Jager et al 2018;Xu et al 2012;Zong et al 2012). Currently, three methods are commonly used for WGA: degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) (Telenius et al 1992), multiple displacement amplification (MDA) (Dean et al 2001;Johne et al 2009), and multiple annealing and looping-based amplification cycles (MALBAC) (Huang et al 2015;Zong et al 2012).…”