Sin-Yu Chen, Yi-Ling Chen, Po-Chen Li, Tai-Shan Cheng, Yeh-Shiu Chu, Yi-Shan Shen, Hsin-Tung Chen, Wei-Ni Tsai, Chien-Ling Huang, Martin Sieber, Yuan-Chieh Yeh, Hsiao-Sheng Liu, Chi-Ling Chiang, Chih-Hung Chang, Andrew S Lee, Yen-Han Tseng, Ly James Lee, Hsiu-Jung Liao, Hon-Kan Yip, Chi-Ying F Huang
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This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI.</p><p><strong>Methods: </strong>A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-β)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI.</p><p><strong>Results: </strong>The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-β-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function.</p><p><strong>Conclusion: </strong>This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. 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引用次数: 0
摘要
背景:急性肺损伤(ALI)是一种危及生命的呼吸系统疾病,其特征是严重的炎症和肺组织损伤,经常导致快速呼吸衰竭和长期并发症。microRNA let-7a-5p 通过调节免疫细胞的活化和细胞因子的产生,参与肺损伤、炎症和纤维化的进展。本研究旨在利用创新的细胞电穿孔平台生成携带let-7a-5p(EV-let-7a-5p)的细胞外囊泡(EVs),EVs来源于转染的沃顿氏果冻-间充质干细胞(WJ-MSCs),作为治疗ALI的潜在基因疗法:方法:采用细胞纳米化(CNP)方法诱导转染了相关质粒DNA的WJ-间充质干细胞产生并释放EV-let-7a-5p。使用切向流过滤(TFF)系统分离条件培养基中的EV-let-7a-5p。EV的表征遵循了国际细胞外囊泡协会(International Society for Extracellular Vesicles)制定的最低共识指南。我们进行了一系列全面的治疗评估,包括使用转化生长因子β(TGF-β)诱导细胞模型的抗纤维化作用、对巨噬细胞极化的调节作用,以及 EV-let-7a-5p 在高氧诱导 ALI 大鼠模型中的影响:结果:CNP平台明显增加了转染WJ-间充质干细胞的EV分泌,工程EV中包裹的let-7a-5p明显高于未处理的WJ-间充质干细胞。这些EV-let-7a-5p不影响细胞增殖,并通过下调LL29细胞中SMAD2/3磷酸化,有效缓解了TGF-β诱导的纤维化表型。此外,EV-let-7a-5p 还能调节炎症微环境中 M2 样巨噬细胞的活化,并显著诱导白细胞介素(IL)-10 的分泌,这证明了它们对炎症的调节作用。在高氧诱导的 ALI 大鼠模型中,给予未经处理的 WJ-间充质干细胞的 EVs 可轻微改善肺功能,并增加血浆中 let-7a-5p 的表达。相比之下,EV-let-7a-5p 能显著减少巨噬细胞浸润和胶原沉积,同时增加 IL-10 的表达,从而大幅改善肺功能:本研究揭示了使用 CNP 平台刺激和转染 WJ-间充质干细胞可产生大量富含 let-7a-5p 的 EVs,这凸显了 EVs 在对抗炎症反应、纤维化活化和高氧诱导的肺损伤方面的治疗潜力。这些结果为开发创新的治疗方法提供了潜在的途径,以更有效地干预 ALI。
Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury.
Background: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI.
Methods: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-β)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI.
Results: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-β-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function.
Conclusion: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.
期刊介绍:
The Journal of Biomedical Science is an open access, peer-reviewed journal that focuses on fundamental and molecular aspects of basic medical sciences. It emphasizes molecular studies of biomedical problems and mechanisms. The National Science and Technology Council (NSTC), Taiwan supports the journal and covers the publication costs for accepted articles. The journal aims to provide an international platform for interdisciplinary discussions and contribute to the advancement of medicine. It benefits both readers and authors by accelerating the dissemination of research information and providing maximum access to scholarly communication. All articles published in the Journal of Biomedical Science are included in various databases such as Biological Abstracts, BIOSIS, CABI, CAS, Citebase, Current contents, DOAJ, Embase, EmBiology, and Global Health, among others.