Neurogenic Effect of Hypoxia-Conditioned UC-MSC Secretome from Macaca fascicularis in an In Vitro Stroke Model

Abstract

Purpose: Mesenchymal stem cells (MSCs) hold therapeutic promise for ischemic stroke through paracrine release of bioactive factors that promote angiogenesis and neurogenesis. This study evaluated the neurogenic potential of Macaca fascicularis UC-MSC secretome under hypoxic and normoxic conditions in an in vitro ischemic stroke model, and assessed its promise as a regenerative, cell-free therapy. Methods: mUC-MSCs were cultured under normoxic (21% O₂) and hypoxic (3% O₂) conditions for 48 hours. Secretomes were collected and analyzed for Brain-Derived Neurotrophic Factor (BDNF) and Stromal Cell-Derived Factor-1 (SDF-1) levels via enzyme-linked immunosorbent assay (ELISA), while exosome morphology was characterized using scanning electron microscopy (SEM). An in vitro ischemic stroke model was created using SH-SY5Y cells exposed to 6 hours of oxygen-glucose deprivation (OGD) followed by 24 hours of reperfusion. During reperfusion, cells were treated with normoxic or hypoxic secretome. Neurogenesis-related gene expression was assessed by RT-PCR and statistically analyzed. Results: The secretome from 3% O2 hypoxic preconditioning showed elevated levels of BDNF and SDF-1 compared to normoxic conditions. RT-qPCR analysis revealed significant upregulation of neurogenesis-related genes BDNF, TrkB, ERK1/2, Nestin, and β-tubulin following treatment with the hypoxic secretome, indicating enhanced neurogenic activity and neuronal survival in the in vitro ischemic stroke model. Conclusion: Hypoxia preconditioned UC-MSC secretome enhances neurogenesis by upregulating key neurogenic markers, promoting neuronal differentiation and regeneration in ischemic conditions. These results highlight the promise of this approach as a regenerative, cell-free therapy for promoting recovery following ischemic stroke.

Keywords: ischemic stroke, neurogenesis, oxygen glucose deprivation (OGD), secretome, SHSY-5Y