De Palma Sara T, Hermans Eva C, Shamorkina Tatiana M, Trayford Chloe, van Rijt Sabine, Heck Albert J R, Nijboer Cora H A, de Theije Caroline G M
Department for Developmental Origins of Disease, University Medical Center Utrecht Brain Center and Wilhelmina Children's Hospital (S.T.D.P., E.C.H., C.H.A.N., C.G.M.d.T.).
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences (T.M.S., A.J.R.H.).
Stroke. 2025 Apr 18. doi: 10.1161/STROKEAHA.124.048964.
BACKGROUND: Neonatal hypoxic-ischemic (HI) brain injury is one of the leading causes of long-term neurological morbidity in newborns. Current treatment options for HI brain injury are limited, but mesenchymal stem cell (MSC) therapy is a promising strategy to boost neuroregeneration after injury. Optimization strategies to further enhance the potential of MSCs are under development. The current study aimed to test the potency of hypoxic preconditioning of MSCs to enhance the therapeutic efficacy in a mouse model of neonatal HI injury. METHODS: HI was induced on postnatal day 9 in C57Bl/6 mouse pups. MSCs were cultured under hypoxic (hypoxic-preconditioned MSCs [HP-MSCs], 1% O) or normoxic-control (normoxic-preconditioned MSCs, 21% O) conditions for 24 hours before use. At 10 days after HI, HP-MSCs, normoxic-preconditioned MSCs, or vehicle were intranasally administered. Gold nanoparticle-labeled MSCs were used to assess MSC migration 24 hours after intranasal administration. At 28 days post-HI, lesion size, sensorimotor outcome, and neuroinflammation were assessed by hematoxylin and eosin staining, cylinder rearing task, and IBA1 staining, respectively. In vitro, the effect of HP-MSCs was studied on transwell migration, neural stem cell differentiation and microglia activation, and the MSC intracellular proteomic content was profiled using quantitative LC-MS/ms. RESULTS: Intranasally administered HP-MSCs were superior to normoxic-preconditioned MSCs in reducing lesion size and sensorimotor impairments post-HI. Moreover, hypoxic preconditioning enhanced MSC migration in an in vitro set-up, and in vivo to the lesioned hemisphere after intranasal application. In addition, HP-MSCs enhanced neural stem cell differentiation into more complex neurons in vitro but had similar anti-inflammatory effects compared with normoxic-preconditioned MSCs. Lastly, hypoxic preconditioning led to elevated abundances of proteins in MSCs related to extracellular matrix remodeling. CONCLUSIONS: This study shows for the first time that hypoxic preconditioning enhanced the therapeutic efficacy of MSC therapy in a mouse model of neonatal HI brain injury by increasing the migratory and neuroregenerative capacity of MSCs.
背景：新生儿缺氧缺血性（HI）脑损伤是新生儿长期神经功能障碍的主要原因之一。目前针对HI脑损伤的治疗选择有限，但间充质干细胞（MSC）疗法是促进损伤后神经再生的一种有前景的策略。进一步提高间充质干细胞潜能的优化策略正在研发中。本研究旨在测试间充质干细胞的缺氧预处理在新生儿HI损伤小鼠模型中增强治疗效果的效力。 方法：在出生后第9天对C57Bl/6小鼠幼崽诱导HI。在使用前，将间充质干细胞在缺氧（缺氧预处理间充质干细胞[HP-MSCs]，1%氧气）或常氧对照（常氧预处理间充质干细胞，21%氧气）条件下培养24小时。HI后10天，经鼻给予HP-MSCs、常氧预处理间充质干细胞或赋形剂。使用金纳米颗粒标记的间充质干细胞评估经鼻给药24小时后的间充质干细胞迁移。HI后第28天，分别通过苏木精和伊红染色、圆筒饲养任务和IBA1染色评估损伤大小、感觉运动结果和神经炎症。在体外，研究了HP-MSCs对Transwell迁移、神经干细胞分化和小胶质细胞激活的影响，并使用定量液相色谱-质谱/质谱对间充质干细胞的细胞内蛋白质组含量进行了分析。 结果：经鼻给予的HP-MSCs在减少HI后损伤大小和感觉运动障碍方面优于常氧预处理间充质干细胞。此外，缺氧预处理在体外实验中增强了间充质干细胞的迁移，并在体内经鼻应用后增强了其向损伤半球的迁移。此外，HP-MSCs在体外增强了神经干细胞向更复杂神经元的分化，但与常氧预处理间充质干细胞相比，具有相似的抗炎作用。最后，缺氧预处理导致间充质干细胞中与细胞外基质重塑相关的蛋白质丰度升高。 结论：本研究首次表明，缺氧预处理通过提高间充质干细胞的迁移和神经再生能力，增强了间充质干细胞疗法在新生儿HI脑损伤小鼠模型中的治疗效果。
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