Labusek Nicole, Mouloud Yanis, Köster Christian, Diesterbeck Eva, Tertel Tobias, Wiek Constanze, Hanenberg Helmut, Horn Peter A, Felderhoff-Müser Ursula, Bendix Ivo, Giebel Bernd, Herz Josephine
Department of Pediatrics I, Neonatology & Experimental Perinatal Neurosciences, Centre for Translational and Behavioral Sciences (C-TNBS), University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Institute for Transfusion Medicine, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Inflamm Regen. 2023 Apr 17;43(1):24. doi: 10.1186/s41232-023-00274-6.
Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia-ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action.
Nine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples.
Intranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression.
Our data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.
人间充质基质细胞（MSC）衍生的细胞外囊泡（EV）在各种脑损伤模型中显示出神经保护潜力，包括缺氧缺血（HI）引起的新生儿脑病。然而，对于MSC-EV疗法的临床转化，需要规模化生产策略，而由于供体间和供体内的异质性，这对原代MSC来说具有挑战性。因此，我们建立了一种克隆扩增并永生化的人MSC系（ciMSC），并在HI诱导的脑损伤小鼠模型中比较了其EV与原代MSC的EV的神经保护潜力。根据其假定的多模式作用机制，全面表征了ciMSC-EV的体内活性。
9日龄C57BL/6小鼠暴露于HI，然后在HI后第1、3和5天重复经鼻递送原代MSC-EV或ciMSC-EV。假手术动物作为健康对照。为了比较两种EV制剂的神经保护作用，在HI后7天通过甲酚紫染色评估全脑和局部脑萎缩。进行免疫组织化学、蛋白质印迹和实时PCR以研究神经炎症和再生过程。通过对血清样本进行多重分析来评估外周炎症介质的量。
经鼻递送ciMSC-EV和原代MSC-EV可同等程度地保护新生小鼠免受HI诱导的脑组织萎缩。从机制上讲，应用ciMSC-EV可减少小胶质细胞活化和星形胶质细胞增生、内皮细胞活化和白细胞浸润。这些作用与脑中促炎细胞因子IL-1β的下调以及抗炎细胞因子IL-4和TGF-β的表达升高有关，而外周血中细胞因子的浓度未受影响。ciMSC-EV介导的脑内抗炎作用伴随着神经祖细胞和内皮细胞增殖增加、少突胶质细胞成熟以及神经营养生长因子表达增加。
我们的数据表明ciMSC-EV通过抑制神经炎症和促进神经再生保留了原代MSC-EV的神经保护作用。由于ciMSC可以克服与MSC异质性相关的挑战，它们似乎是规模化生产基于EV的疗法以治疗新生儿以及可能还有成人大脑损伤的理想细胞来源。
