Hematopoietic stem cells (HSCs) within the bone marrow (BM) display significant molecular and functional heterogeneity. Deciphering intrinsic factors that govern HSC diversity is key to enriching specific HSC subtypes for predictable and clinically relevant differentiation outcomes. Here, we show that the mitochondrial protein Asrij/OCIAD1, a conserved regulator of hematopoietic homeostasis, contributes to HSC heterogeneity. Asrij depletion is known to cause loss of quiescence, myeloid bias, and aging-like changes in mouse BM HSCs. Interestingly, Asrij expression is inherently heterogeneous and enriched in only 47% of the HSC population. To investigate whether Asrij expression levels influence HSC fate, we generated a novel Asrij-mNeonGreen (mNG) knock-in reporter mouse using CRISPR-Cas9 technology. We show that the Asrij reporter faithfully recapitulates its heterogeneous expression in the BM HSCs, allowing isolation of live cells based on Asrij expression levels. Ex vivo culture of HSCs demonstrated that AsrijHSCs exhibit enhanced self-renewal capacity, whereas AsrijHSCs are primed for differentiation. Transplantation assays further revealed that AsrijHSCs have enhanced reconstitution in the BM hematopoietic stem and progenitor cell (HSPC) and myeloid cell compartments. Transcriptomic analysis uncovered signatures of quiescence in AsrijHSCs, whereas AsrijHSCs exhibit hallmarks of HSC activation. In summary, we show that Asrij levels impact the quiescence, self-renewal, and differentiation potential of HSCs, thereby contributing to the functional diversity of the HSC pool. Furthermore, the Asrij-mNG reporter mouse provides a powerful and versatile model for investigating the molecular underpinnings of functional diversity within the HSC compartment. low high low low high
