Transcriptional heterogeneity in hematopoietic stem cells prospectively predicts clonal fitness in aged microenvironments

Cancers are diseases of aging. Aging alters hematopoietic stem cell (HSC) function and clonal composition, yet whether intrinsic transcriptional heterogeneity at baseline determines which clones expand or persist in aged niches remains unknown. Dominant clones often emerge during aging even without clonal hematopoiesis (CH) driver mutations, indicating that phenotypic variation alone can reshape clonal competition. This gap limits our ability to predict or modulate age-related clonal imbalance, CH, and leukemia risk. We hypothesize that aging amplifies heritable phenotypic variation in HSC, enhancing cellular heterogeneity that drives leukemic transformation. We used CellTag Indexing and scRNA-seq to longitudinally track the fate of shared mouse HSC sister clones across heterochronic and homochronic transplantation, enabling direct linkage of pre-transplant transcriptional states to post-transplant clonal fitness. Independent experiments validated that simultaneous single-cell lineage and transcriptome sequencing maps functional HSC heterogeneity in vivo . We quantified (i) intra- and inter-cellular transcriptional heterogeneity at baseline (Day 0 pre-transplant), (ii) clone-specific self-renewal and expansion at Day 60 post-transplant, and (iii) clone-wise linkage between baseline transcriptome programs and fitness using predictive AI models with 5-fold cross validation. We found that a significant increase in transcriptomic heterogeneity in long-term HSC (LT-HSC) from old mice was primarily driven by the donor age rather than the host age. LT-HSC from old donor mice (20 mon) are enriched for up-regulated differential variable genes (DVGs) compared to young donor mice (2 mon) in regulation of cell cycle, myeloid differentiation, and mitochondrial regulation, which is validated by unmanipulated HSC scRNA-seq data. Notably, sister clones derived from aged donors exhibited significantly higher self-renewal and clonal fitness in aged versus young recipients, indicating selective compatibility between intrinsic aging programs and niche age. Furthermore, larger (positively selected) clones at Day 60 compared to smaller clones exhibit higher pre-existing (Day 0) HSC intra-clonal heterogeneity. Day 0 and 60 heterogeneity both robustly predicted long-term self-renewal across transplantation contexts. Clones achieving high fitness in aged hosts were prospectively predicted, prior to transplantation, by up-regulated transcriptional signatures of regulator of self-renewal, cell cycle and stress conditions, and inflammation by random forest model feature importance ranking. The human orthologues of these gene are significantly prognostic for overall survival among acute myeloid leukemia patients (TCGA), underscoring its translational relevance. These results support that aging encodes clone-specific, cell-intrinsic “starting states” that interact with microenvironmental age to shape hematopoietic output over time. By prospectively linking pre-existing HSC state variation to subsequent clonal fitness, our study extends the current understanding of evolutionary trajectories of HSC aging and nominates actionable molecular programs that can be targeted to rebalance hematopoiesis and reduce malignant evolution in aging populations.