EXTRACELLULAR MIR-25/MIR-106A RELEASED BY HYPOXIC HUMAN NEURONS AND ASTROCYTES ACTIVATES NEUROGENESIS-RELATED PATHWAYS IN CONDITIONED NPCS

Stroke is the second leading cause of death worldwide, and ischemic stroke is the most common type, accounting for 84% of cases. Ischemic injury may promote endogenous repair mechanisms, including neurogenesis and neuroplasticity, which are regulated by microRNAs (miRNAs) and guidance molecules. Ischemic-like hypoxia reshapes intercellular communication within the human neural niche, but the contribution of extracellular miRNA remains incompletely defined. We generated neurons and astrocytes from human induced pluripotent stem cell-derived neural progenitor cells (NPCs) and exposed them to hypoxia followed by reoxygenation. We quantified miR-25-3p and miR-106a in culture media and assessed whether conditioned media from hypoxic and reoxygenated cells modulate neurogenesis-related pathways in naïve NPCs.
Under hypoxia/reoxygenation, both neurons and astrocytes showed a significant increase in extracellular miR-25-3p and miR-106a, indicating active release. NPCs exposed to miRNA-enriched conditioned media displayed selective regulation of specific targets and pathways: HMGB1 expression was significantly reduced, most notably after reoxygenation, whereas p57 (CDKN1C) levels were significantly increased, consistent with miRNA-mediated post-transcriptional control of cell-cycle and differentiation programs. In parallel, BDNF levels were markedly increased in the culture media of NPCs exposed to reoxygenated conditioned media, indicating enhanced paracrine trophic signaling. These findings identify a hypoxia-induced extracellular miRNA-based communication mechanism whereby neurons and astrocytes influence NPC signaling through miR-25- and miR-106a-dependent regulation of neurogenesis-related pathways.