Single-cell architecture of purinergic signaling in human cord blood hematopoietic stem and progenitor cells

Purinergic signaling has emerged as a key regulator of hematopoietic stem and progenitor cell (HSPC) trafficking, metabolism, and innate immune responsiveness. Our previous studies demonstrated that extracellular ATP promotes HSPC mobilization, homing, and engraftment by activating P2X purinergic receptors and engaging the Nlrp3 inflammasome downstream, whereas enzymatic conversion of ATP to extracellular adenosine exerts opposite, anti-inflammatory effects. Subsequently, we postulated that purinergic signaling is an evolutionarily ancient regulatory system that remains intrinsically embedded within the hematopoietic stem cell program. However, its transcriptional organization across distinct human HSPC subsets remains unknown. We applied single-cell RNA sequencing to human umbilical cord blood-derived CD133⁺Lin⁻CD45⁺ and CD34⁺Lin⁻CD45⁺ cells enriched for HSPCs. We identified transcriptionally distinct clusters representing primitive progenitors and lineage-primed intermediates, and we demonstrated a hierarchical organization of purinergic receptors and nucleotide-metabolizing enzymes across these populations. Primitive HSPCs exhibited a restricted purinergic repertoire coupled with intracellular nucleotide recycling machinery, consistent with a tightly regulated metabolic-immune state. Lineage-biased clusters showed selective enrichment of receptors and ectonucleotidases associated with inflammatory activation, migration, and fate commitment. Together, these findings establish purinergic signaling as a fundamental, cell-intrinsic regulator of early hematopoiesis and highlight how this ancient signaling pathway shapes human stem cell fate decisions.