Single-cell analysis reveals different age-related somatic mutation profiles between stem and differentiated cells in human liver

Abstract

Accumulating somatic mutations have been implicated in age-related cellular degeneration and death. Because of their random nature and low abundance, somatic mutations are difficult to detect except in single cells or clonal lineages. Here we show that in single hepatocytes from human liver, an organ normally exposed to high levels of genotoxic stress, somatic mutation frequencies are high and increase substantially with age. Significantly lower mutation frequencies were observed in liver stem cells and organoids derived from them. These results could explain the increased age-related incidence of liver disease in humans and stress the importance of stem cells in maintaining genome integrity.Genome integrity is critically important for cellular function. Evidence has accumulated that loss of genome integrity and the increasingly frequent appearance of various forms of genome instability, from chromosomal aneuploidy to base substitution mutations, is a hallmark of aging (1, 2). However, somatic mutations occur de novo across cells and tissues and are of low abundance. Hence, thus far, of all mutation types only chromosomal alterations could readily be studied directly during in vivo aging using cytogenetic methods (3). More recently it has become possible to also study low-abundant somatic mutations by whole genome sequencing (WGS) of single cells or clones derived from single cells (4-7). Such studies confirmed that like chromosomal alterations also base substitution mutations accumulate with age in human brain (8,9), intestine and liver (4).Due to its high metabolic and detoxification activity liver is more likely to be adversely affected by various damaging agents than most other organs. In humans this may cause agerelated loss of function, most notably a severe reduction in metabolic capacity and multiple pathologies, including fatty liver disease, cirrhosis, hepatitis, infections and cancer (10,11). To test if somatic mutations occur with a high frequency and accumulate rapidly with age we performed WGS on single primary hepatocytes from human donors varying in age between 5 months and 77 years of age. These cells were isolated from healthy human individuals shortly after death through perfusion of whole donor livers (Lonza Walkersville Inc.). Cell viability was higher than 80% and, after staining with Hoechst, hepatocytes were isolated via FACS into individual PCR tubes as diploid single cells (Fig. S1). In total we sequenced 4 single hepatocytes and bulk genomic liver DNA for each of eight human donors (Table S1). Each cell was subjected to our recently developed procedure for whole genome amplification (WGA) and sequencing (5,6). Somatic single nucleotide variants (SNVs) in single cells were identified relative to bulk genomic DNAs at a depth of ≥ 20X using a combination of VarScan2, MuTect2 and Haplotypecaller software with certain modifications (Methods , Table S2). Overlapping mutations, revealed by all three analytical tools, were exclusively considered for further analysis.After adjusting for genomic ...

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“…Interestingly, the rate of accumulation of SNVs increased with age, while the mutational signatures observed in B cell cancers were elevated in the cells from older donors. A similar observation was reported in a scDNA-seq analysis of stem and differentiated liver cells, where frequency of somatic mutations increased significantly in differentiated hepatocytes with age across heterogenous single-cell populations [31].…”

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“…4F). However, this may still be an overestimate of the true number, as our less stringent variant filtering criteria resulted in a higher number of variants per cell than some other estimates in newborn cells (24)(25)(26). Taken together, these data confirm that PTA can accurately detect a much larger number of germline variants across the genome of single cells than existing WGA methods with high precision and that the higher quality variants can be used to estimate per-cell somatic mutation rates.…”

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“…Moreover, by its very nature, this approach exclusively analyzes mutations in stem or progenitor cells, which generally have a lower mutation rate than differentiated cells. This is best illustrated by a recent direct comparison of human liver stem cells with fully differentiated hepatocytes, showing an approximately 2-fold higher average mutation frequency in the latter (Brazhnik et al, 2020). In addition, fully differentiated cells also displayed a much higher cellto-cell variation in mutation frequencies, which is probably a reflection of their position in the hierarchy of cellular differentiation.…”