Serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of mesenchymal stem cells on experimental renal fibrosis

Abstract

Background: Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia-reperfusion injury (IRI).

Methods: SF-MSCs were incubated under 1% O₂ conditions (hypo-SF-MSCs) or 21% O₂ conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs.

Results: Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling.

Conclusions: These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Keywords: Hepatocyte growth factor; Hypoxic preconditioning; Mesenchymal stem cells; Renal fibrosis; Serum-free conditions.