Hematopoietic stem cells undergo bidirectional fate transitions in vivo

Differentiation of haematopoietic stem and progenitor cells (HSPCs) is widely considered unidirectional in vivo. Here, we developed clonal phylogenetic tracing (CP-tracer) by sequential genetic barcoding, which enabled high-resolution analysis of 181,695 subclones derived from 847 individually labelled HSPCs. This approach uncovered bidirectional fate transitions between myeloid-biased (My-) and lineage-balanced haematopoietic stem cells (HSCs). Individual HSC clones, distinguished by temporally unique serial barcodes, exhibited durable lineage bidirectionality, with dynamics favouring progressive accumulation of My-HSCs over time. CRISPR-Cas9 screening identified the homeobox gene Hhex as a suppressor of myeloid differentiation that enables pivoting toward balanced output. Hhex function is age-dependent, with its expression declining in aged HSCs. Together, these findings demonstrate unexpected plasticity in HSC differentiation, modulated in part by Hhex-mediated repression, that shifts over time to drive the myeloid bias characteristic of ageing.