Background and Purpose: Human mesenchymal stem cells (MSCs) show promise for treating ischaemic and inflammatory conditions, yet their clinical utility is limited by a failure to replicate preclinical efficacy. We hypothesized that dual priming of human bone marrow-derived MSCs (hBMSCs) with interleukin-1alpha (IL-1α) and cobalt chloride, CoCl 2 (a hypoxia mimetic) would potentiate their therapeutic efficacy for hypoxic-ischaemic conditions. Experimental Approach: hBMSCs were primed individually or in combination. Their conditioned media (CM) was analysed for relevant secreted biomolecules. To test the anti-inflammatory and cytoprotective properties, primed-hBMSCs-derived CM was applied to LPS-treated BV2 microglial cells ( in vitro inflammation model) and BV2 cells exposed to oxygen glucose deprivation (OGD) ( in vitro hypoxic injury model). The neuroprotective effect was evaluated by assessing neuronal death (using NLCs) following exposure to OGD/reoxygenation (OGD/R) in the presence of various CMs. Key Finding: Priming significantly altered hBMSCs’ secretion profiles. IL-1α triggered significant ( p<0.05) release of inflammatory mediators/cytokines, notably TNF-α, ICAM-1, and G-CSF. Except for G-SCF, dual-priming did not potentiate the release of these inflammatory molecules. However, while single priming with either IL-1α or hypoxia did not induce remarkable secretion of the neurotrophins β-NGF and BDNF, the dual-priming resulted in significant ( p<0.05) and additive secretion of these neurotrophins. In the LPS model, CM from dual-primed hBMSCs significantly reduced the secretion of pro-inflammatory cytokines, TNF-α and IL-6. In the hypoxia model, priming did not significantly influence the marked reduction ( p<0.05) of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Lastly, CMs from primed hBMSCs significantly (p<0.05) reduced neuronal cytotoxicity, yet only CM from dual-primed hBMSCs significantly (p<0.05) preserved the viability of NLCs following 24 h reperfusion after OGD. Conclusion: Dual priming of hBMSCs with both hypoxia and IL-1α significantly enhanced their therapeutic potential for managing hypoxic-ischemic conditions and offers a promising strategy for developing more effective cell-based therapies for ischaemic stroke.

Background and Purpose: Human mesenchymal stem cells (MSCs) show promise for treating ischaemic and inflammatory conditions. However, their clinical utility is limited by issues like poor migration, engraftment, rapid elimination, and slow functional recovery post-thaw. We investigated whether dual priming of human bone marrow-derived MSCs (hBMSCs) with IL-1α and CoCl2 will enhance their therapeutic potential. Experimental Approach: hBMSCs were primed individually or in combination with IL-1α and CoCl2. Their conditioned medium (CM) was then analysed for secreted biomolecules, including G-CSF, IL-8, BDNF, β-NGF, ICAM-1, and TNF-α. Two in vitro models using BV2 cells were employed: one to assess anti-inflammatory efficacy by treating LPS-induced inflammation with CM, and another to evaluate cytoprotective effects by exposing cells to oxygen and glucose deprivation/reperfusion in the presence of CM. Markers of inflammation, neuroinflammation, and cell death were subsequently measured. Key Finding: Priming significantly altered hBMSCs secretion profiles: IL-8 decreased (~3-fold), while β-NGF (1.3-1.9-fold) and ICAM-1 (2.7-4-fold) increased. G-CSF (100-400 pg/ml) and BDNF (~165 pg/ml) became detectable. TNF-α response varied, decreasing with IL-1α priming but increasing with dual priming. In both LPS-induced inflammation and OGD/reperfusion models, CM from primed hBMSCs significantly reduced IL-6, IL-1β, enhanced IL-10, and decreased MMP-9 expression. Notably, while all hBMSCs CMs alleviated LPS-induced cell death, only dual-primed CM demonstrated significant cytoprotective effects in the ischaemia/reperfusion model. Conclusion: Dual priming of hBMSCs with both hypoxia (CoCl2 and IL-1α significantly enhanced their therapeutic potential for managing hypoxic-ischaemic conditions and offers a promising strategy for developing more effective cell-based therapies for Ischaemic stroke