Our results so far indicate that endogenous aldehydes give rise to double-stranded breaks in the absence of Fanconi anaemia repair, which are engaged by homologous recombination and NHEJ, but ultimately rearrange chromosomes in bone marrow cells. A key question is whether endogenous DNA damage and subsequent mutations accumulate in the HSC compartment. This is a critical question because there is evidence that HSCs differ in their DNA repair capacity and response compared to later progenitors11. Two obstacles had to be overcome in order to establish whether endogenous aldehydes mutate the genomes of these vital cells. First, the stochastic nature of DNA damage makes it unlikely that the same mutation will occur in multiple cells. Second, the scarcity of HSCs, especially in the case of Aldh2−/−Fancd2−/− mice, precludes the use of most conventional techniques to assess DNA damage. We also wanted to ascertain whether mutations arise in functional stem cells, and therefore avoided whole-genome amplification or short-term in vitro expansion of cells isolated by flow cytometry. Instead, we decided to define HSCs functionally and exploit the ability of a single HSC to reconstitute long-term blood production following transplantation into a lethally irradiated mouse20.
