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

中文翻译：

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无血清培养基和缺氧预处理协同增强间充质干细胞对实验性肾纤维化的治疗作用

间充质干细胞 (MSC) 以旁分泌方式修复受损组织。为了增强它们的治疗特性，已经研究了各种因素的预处理。我们之前已经表明，在无血清培养基 (SF-MSCs) 中培养的 MSCs 可促进其免疫抑制能力，从而增强其抗纤维化作用。在这里，我们检查了无血清培养基和缺氧预处理是否协同增强了 MSCs 对缺血再灌注损伤（IRI）大鼠肾纤维化的治疗作用。SF-MSCs 在收集前在 1% O2 条件 (hypo-SF-MSCs) 或 21% O2 条件 (normo-SF-MSCs) 下孵育 24 小时。IRI 手术后，通过腹主动脉注射低 SF-MSC 或正常 SF-MSC。在注射后 7 或 21 天，杀死大鼠并收集它们的肾脏以评估炎症和纤维化。在体外实验中，我们使用转化生长因子 (TGF)-β1 刺激的 HK-2 细胞研究了低 SF-MSC 是否增强了抗纤维化体液因子的分泌，这些细胞与来自低 SF-MSC 或标准 SF 的条件培养基一起孵育-MSC。Normo-SF-MSCs 显示衰老减弱，这增加了它们的增殖能力。尽管在normo-SF-MSCs和hypo-SF-MSCs之间没有发现细胞衰老的显着差异，但与normo-SF-MSCs相比，hypo-SF-MSCs进一步增加了它们的增殖能力。此外，给予hypo-SF-MSCs比给予normo-SF-MSCs更强烈地改善肾纤维化。而且，尽管与用 PBS 治疗的对照大鼠相比，hypo-SF-MSC 强烈减弱了炎症细胞的浸润，但这种减弱在正常 SF-MSC 和低 SF-MSC 之间几乎相等。体外实验表明，与正常-SF-MSCs 相比，hypo-SF-MSCs 更显着地抑制转化生长因子 (TGF)-β/Smad 信号传导。此外，即使在无血清条件下，缺氧预处理也会增加肝细胞生长因子 (HGF) 的分泌，而在低 SF-MSC 中敲除 HGF 会减弱对 TGF-β/Smad 信号的抑制。这些结果表明，施用离体扩增的、缺氧预处理的 SF-MSC 可能是预防肾纤维化的有用细胞疗法。体外实验表明，与正常-SF-MSCs 相比，hypo-SF-MSCs 更显着地抑制转化生长因子 (TGF)-β/Smad 信号传导。此外，即使在无血清条件下，缺氧预处理也会增加肝细胞生长因子 (HGF) 的分泌，而在低 SF-MSC 中敲除 HGF 会减弱对 TGF-β/Smad 信号的抑制。这些结果表明，施用离体扩增的、缺氧预处理的 SF-MSC 可能是预防肾纤维化的有用细胞疗法。体外实验表明，与正常-SF-MSCs 相比，hypo-SF-MSCs 更显着地抑制转化生长因子 (TGF)-β/Smad 信号传导。此外，即使在无血清条件下，缺氧预处理也会增加肝细胞生长因子 (HGF) 的分泌，而在低 SF-MSC 中敲除 HGF 会减弱对 TGF-β/Smad 信号的抑制。这些结果表明，施用离体扩增的、缺氧预处理的 SF-MSC 可能是预防肾纤维化的有用细胞疗法。