The human bone marrow constitutes the principal site of hematopoiesis, sustaining the continuous generation of platelets, erythrocytes and immune cells through the activity of hematopoietic stem and progenitor cells (HSPCs) residing within specialized microenvironmental niches. Within this compartment, a rare subset of hematopoietic stem cells (HSCs) maintains lifelong hematopoiesis by precisely regulating the balance between self-renewal and differentiation, predominantly persisting in a quiescent state under homeostatic conditions but capable of rapid activation in response to acute physiological stressors such as infection or hemorrhage. The equilibrium between steady-state and emergency hematopoiesis is fundamental to hematopoietic homeostasis; however, chronic inflammation and aging can disrupt this balance, leading to impaired blood cell production and increased risk of developing hematologic malignancies. Although substantial progress has been made in the characterization and prospective isolation of distinct human HSPC subsets using surface marker-based strategies, current strategies often yield in heterogeneous populations and several differentiation models have been proposed. To address this challenge and map differentiation trajectories, the presented research employed a single cell Transcriptomic/AbSeq approach, simultaneously quantifying the expression of 596 genes at mRNA level and 46 surface markers, on over 62,000 FACS-enriched HSPCs from 15 healthy donors across different age groups. Comprehensive computational analysis revealed four main lineage pathways, supporting a stepwise model of differentiation with an early branching point for megakaryocyte-erythroid progenitors. Notably, HSPCs from older donors exhibited a higher proportion of undifferentiated cells and diminished differentiation across all lineages. A key finding of this study is the identification of Programmed death ligand 2 (PD-L2/CD273) as a surface marker highly expressed on the most primitive HSPCs. CD273/PD-L2-positive HSPCs, isolated from mobilized PB samples, demonstrated a distinct molecular signature characterized by the enrichment of stemness genes such as Thy1, DLK1 and MPL, as well as delayed entry into the cell cycle, reduced mitochondrial activity and delayed in vitro differentiation indicating a deeper quiescent state. Functional assays, including in vitro colony forming assays and in vivo xenograft experiments, confirmed that CD273/PD-L2high HSPCs possess the capacity for multi-lineage reconstitution. Beyond their stem cell properties, CD273/PD-L2-expressing HSPCs exhibited notable immunomodulatory functions. In allogeneic lymphocyte reaction assays, these cells suppressed CD8+ T-cell proliferation and activation. Blocking PD-L2 promoted the secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6), showed a reduced abundance of regulatory T-cells and a shift toward myeloid lineage bias in HSPCs, implying an increased inflammatory response reaction. These findings highlight a dual role for PD-L2: First as a marker of more quiescent, primitive HSPCs and second as a mediator of immune regulation within the hematopoietic compartment.
In summary, this integrated study provides valuable insights into the organization of human hematopoiesis and the identification of PD-L2/CD273 as a defining marker of a particularly quiescent, immunomodulatory HSPC subset. These discoveries may have important implications for stem cell transplantation and the treatment of blood malignancies, as understanding and manipulating these pathways may improve therapeutic outcomes and help maintaining healthy hematopoiesis throughout life.
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