Asrij/OCIAD1 expression delineates functionally distinct hematopoietic stem cells in the bone marrow.

Abstract

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 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 Asrij^low HSCs exhibit enhanced self-renewal capacity, whereas Asrij^high HSCs are primed for differentiation. Transplantation assays further revealed that Asrij^low HSCs have enhanced reconstitution in the BM hematopoietic stem and progenitor cell (HSPC) and myeloid cell compartments. Transcriptomic analysis uncovered signatures of quiescence in Asrij^low HSCs, while Asrij^high HSCs 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. Further, the Asrij-mNeonGreen reporter mouse provides a powerful and versatile model for investigating the molecular underpinnings of functional diversity within the HSC compartment. TEASER ABSTRACT: Our study addresses the complexities of hematopoietic stem cell (HSC) heterogeneity and uncovers novel determinants that govern long-term (LT) reconstituting HSC function. Using a novel Asrij-mNeonGreen fluorescent reporter mouse, we show that the mitochondrial protein Asrij/OCIAD1 regulates HSC heterogeneity. We show that low Asrij expression marks quiescent HSCs with robust self-renewal capacity, whereas high Asrij expression identifies activated, differentiation-primed HSCs. These findings position Asrij as a novel determinant of HSC heterogeneity and introduce the Asrij-mNeonGreen reporter as a versatile tool for dissecting stem cell fate decisions in-vivo.