Hematopoietic stem cells undergo bidirectional fate transitions in vivo.

Transitions between subsets of differentiating hematopoietic cells are widely regarded as unidirectional in vivo. Here, we introduce clonal phylogenetic tracer (CP-tracer) that sequentially introduces genetic barcodes, enabling high-resolution analysis of [~]100,000 subclones derived from [~]500 individual hematopoietic stem cells (HSC). This revealed previously uncharacterized HSC functional subsets and identified bidirectional fate transitions between myeloid-biased and lineage-balanced HSC. Contrary to the prevailing view that the more self-renewing My-HSCs unidirectionally transition to balanced-HSCs, phylogenetic tracing revealed durable lineage bidirectionality with the transition favoring My-HSC accumulation over time1,2. Further, balanced-HSCs mature through distinct intermediates--My-HSCs and lymphoid-biased-HSCs--with lymphoid competence here shown by CRISPR/Cas9 screening to be dependent on the homeobox gene, Hhex. Hhex enables Ly-HSC differentiation, but its expression declines with age. These findings establish HSC plasticity and Hhex as a determinant of myeloid-lymphoid balance with each changing over time to favor the age-related myeloid bias of the elderly. HighlightsO_LISequenctial introduction of DNA barcodes in vivo was developed to assess time dependent changes in cell fate. C_LIO_LIClonal phylogenetic tracer (CP-tracer) enabled high-resolution phylogenetic analysis of [~]100,000 subclones derived from [~]500 individual hematopoietic stem cells (HSC). C_LIO_LIBidirectional fate transitions between myeloid-biased haematopoietic stem cells (My-HSCs) and lineage-balanced haematopoietic stem cells (balanced-HSCs) were observed. C_LIO_LIHhex was identified as a molecular driver of HSC lymphoid competence. C_LI