Thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles attenuate experimental necrotizing enterocolitis.

IF 7.3 2区医学 Q1 CELL & TISSUE ENGINEERING Stem Cell Research & Therapy Pub Date : 2025-02-28 DOI:10.1186/s13287-025-04243-3

Sein Hwang, Se In Sung, Young Eun Kim, Misun Yang, Ara Koh, So Yoon Ahn, Yun Sil Chang

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Abstract

Background: Necrotizing enterocolitis (NEC) is a critical gastrointestinal disease in preterm infants, for which no specific treatment is established. We previously demonstrated that thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles (thMSC-EVs) enhance protection against other neonatal tissue injuries. Therefore, this study aimed to evaluate the therapeutic potential of thMSC-EVs in modified in vitro, in vivo, and organoid models of NEC.

Methods: In vitro, the effects of thMSC-EVs and naïveMSC-EVs were compared in hyperosmotic, ischemic, and hypothermic (HIT)-stressed IEC-6 cells and LPS-treated peritoneal macrophages. In vivo, NEC was induced in P4 mouse pups by three cycles of formula feeding, oral LPS administration, hypoxia, and hypothermia, followed by overnight dam care. 2 × 10⁹ thMSC-EVs were intraperitoneally administered daily for three days, and the therapeutic effects were assessed macroscopically, histologically, and biochemically. NEC mouse-derived organoids were established to evaluate the thMSC-EVs' effect in mature enterocytes. LC-MS/MS was performed to analyze the EV proteomics.

Results: In vitro, compared with naïveMSC-EVs, thMSC-EVs significantly improved cellular viability in HIT-induced IEC-6 cells and reduced pro-inflammatory (IL-1α, IL-1β, TNF-α) but increased anti-inflammatory (TGF-b) cytokine levels in LPS-treated peritoneal macrophages. In vivo, thMSC-EVs significantly attenuated clinical symptoms, reduced intestinal damage, and retained intestinal stem cell markers, showing more significant localization in NEC-induced intestines than in healthy intestines. In NEC mouse-derived organoids, thMSC-EVs significantly increased OLFM4 and claudin-4 expression and reduced stress-related markers such as sucrase-isomaltase, defensin, and chromogranin A. Proteomic analysis revealed that thMSC-EVs were greater enriched in anti-apoptotic, anti-inflammatory, cell adhesion, and Wnt signaling pathways than naïveMSC-EVs.

Conclusion: thMSC-EVs improved cellular viability, reduced apoptosis, attenuated inflammation, and upregulated key intestinal stem cell markers, collectively suggesting their tissue-protective effects and highlighting their potential as a treatment for NEC.

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凝血酶预处理间充质间质细胞来源的细胞外囊泡减弱实验性坏死性小肠结肠炎。

背景：坏死性小肠结肠炎（NEC坏死性小肠结肠炎（NEC）是早产儿的一种严重胃肠道疾病，目前尚无特效疗法。我们以前曾证实，凝血酶预处理间充质基质细胞衍生的细胞外囊泡（thMSC-EVs）能增强对其他新生儿组织损伤的保护。因此，本研究旨在评估thMSC-EVs在改良的NEC体外、体内和类器官模型中的治疗潜力：方法：在体外，比较了thMSC-EVs和naïveMSC-EVs在高渗、缺血和低体温（HIT）应激的IEC-6细胞和LPS处理的腹腔巨噬细胞中的作用。在体内，通过配方喂养、口服 LPS、缺氧和低体温三个周期诱导 P4 小鼠幼崽发生 NEC，然后对幼崽进行隔夜护理。每天腹腔注射 2 × 109 thMSC-EV，连续三天，并从宏观、组织学和生化角度评估治疗效果。为了评估thMSC-EVs在成熟肠细胞中的作用，建立了NEC小鼠衍生器官组织。LC-MS/MS分析了EV的蛋白质组学：结果：在体外，与天真MSC-EVs相比，thMSC-EVs能显著提高HIT诱导的IEC-6细胞的活力，降低LPS处理的腹腔巨噬细胞的促炎细胞因子（IL-1α、IL-1β、TNF-α）水平，但增加抗炎细胞因子（TGF-b）水平。在体内，thMSC-EVs能明显减轻临床症状，减少肠道损伤，并保留肠道干细胞标志物，在NEC诱导的肠道中的定位比在健康肠道中更明显。在NEC小鼠衍生的器官组织中，thMSC-EVs显著增加了OLFM4和claudin-4的表达，减少了应激相关标记物，如蔗糖异麦芽糖酶、防御素和chromogranin A。蛋白质组分析表明，与天真MSC-EVs相比，thMSC-EVs更富含抗凋亡、抗炎、细胞粘附和Wnt信号通路。结论：thMSC-EVs提高了细胞活力，减少了细胞凋亡，减轻了炎症反应，并上调了关键的肠道干细胞标志物。

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来源期刊

Stem Cell Research & Therapy CELL BIOLOGY-MEDICINE, RESEARCH & EXPERIMENTAL

CiteScore

13.20

自引率

8.00%

发文量

525

审稿时长

1 months

期刊介绍： Stem Cell Research & Therapy serves as a leading platform for translational research in stem cell therapies. This international, peer-reviewed journal publishes high-quality open-access research articles, with a focus on basic, translational, and clinical research in stem cell therapeutics and regenerative therapies. Coverage includes animal models and clinical trials. Additionally, the journal offers reviews, viewpoints, commentaries, and reports.