Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age^1,2. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation2.Whilemanydrivers of HSC ageing have been proposed ^2-5, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolarassociated cH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoingDNAdamage. Persistent nucleolar cH2AXalso acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis inquiescent oldHSCs.Our results identify replication stress as a potent driver of functional decline inoldHSCs, and highlight theMCMDNAhelicase as a potential molecular target for rejuvenation therapies.