Background: Heart transplantation remains the gold standard treatment for end-stage heart failure. However, the limited availability of donor hearts represents a significant bottleneck. Donation after Circulatory Death (DCD) may expand the donor pool, but it is hindered by prolonged warm ischemia and Ischemia-Reperfusion Injury (IRI), especially in Italy where the functional warm ischemic time is longer than 45 minutes. Since May 2023 60 DCD heart transplant have been performed with a mortality rate of 18%. Innovative strategies for myocardial protection are therefore urgently needed. Aim: This study investigates the protective effects of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in an in vitro model of hypoxia-induced cardiac injury, aiming to explore their potential application in cardiac tissue regeneration and organ preservation for transplantation. Methods: Cardiomyocytes (CMs) were obtained through a 30-day differentiation protocol from human induced pluripotent stem cells (hiPSCs). Morphological and immunohistochemical analyses confirmed the successful differentiation. MSC-EVs were isolated from human umbilical cord-derived mesenchymal stem cells and characterized using Tunable Resistive Pulse Sensing (TRPS) with the Exoid platform. To simulate oxidative injury, hiPSC-derived CMs were exposed to hydrogen peroxide (H₂O₂) for 24 hours. Three experimental groups were analysed via calcium imaging: untreated control CMs, CMs treated with H₂O₂ only, and CMs pre-treated with MSC-EVs before H₂O₂ exposure. Results: Control hiPSC-CMs exhibited synchronized, spontaneous contractions with regular calcium transients and high sarcoplasmic calcium efflux. H₂O₂ treatment significantly impaired contractility, reducing beat frequency and altering calcium kinetics. Pre-treatment with MSC-EVs resulted in partial recovery of physiological calcium dynamics, with increased beat frequency (~15 bpm vs ~5 bpm in H₂O₂-only group) and improved calcium efflux, resembling the control group. Conclusion: MSC-derived EVs showed a promising cardioprotective effect against oxidative stress in vitro, suggesting their potential as therapeutic agents in cardiac graft preservation strategies. These findings support further investigation into EV-based interventions within ex vivo perfusion platforms and cardiac regenerative medicine, particularly in the context of DCD heart transplantation.
Background: Il trapianto cardiaco rappresenta tutt’oggi il trattamento di riferimento per l’insufficienza cardiaca in stadio terminale. Tuttavia, la disponibilità limitata di cuori da donatore costituisce un ostacolo significativo. La donazione a cuore fermo (Donation after Circulatory Death, DCD) potrebbe ampliare il numero di donatori, ma risente dell’effetto negativo dell’ischemia calda prolungata e del danno da ischemia-riperfusione (Ischemia-Riperfusion Injury, IRI), specialmente in Italia dove il tempo di ischemia funzionale calda è superiore ai 45 minuti. Da Maggio 2023 sono stati eseguiti 60 trapianti di cuore DCD con una mortalità del 18%. Diventa quindi fondamentale sviluppare nuove strategie per la protezione miocardica. Obiettivo: Valutare l’effetto protettivo delle vescicole extracellulari derivate da cellule mesenchimali (MSC-EVs) in un modello in vitro di danno ipossico, con l’obiettivo di esplorarne il potenziale applicativo nella rigenerazione tissutale cardiaca e nella conservazione d’organo ai fini del trapianto. Materiali e Metodi: I cardiomiociti (CMs) sono stati ottenuti tramite un protocollo di differenziazione di 30 giorni da cellule staminali pluripotenti indotte umane (hiPSC). La differenziazione è stata confermata attraverso analisi morfologiche e immunoistochimiche. Le MSC-EVs sono state isolate da cellule mesenchimali da cordone ombelicale umano e caratterizzate mediante Tunable Resistive Pulse Sensing (TRPS) con sistema Exoid. Per simulare il danno ossidativo, i CMs sono stati esposti a perossido di idrogeno (H₂O₂) per 24 ore. Sono stati analizzati tre gruppi sperimentali tramite calcium imaging: CMs non trattati (controllo), CMs trattati solo con H₂O₂ e CMs pretrattati con MSC-EVs prima dell’esposizione a H₂O₂. Risultati: I CMs del gruppo controllo mostravano contrazioni spontanee e sincronizzate, con transitori di calcio regolari ed efficiente rilascio di calcio dal reticolo sarcoplasmatico. Il trattamento con H₂O₂ ha compromesso la contrattilità, riducendo la frequenza dei battiti e alterando la cinetica del calcio intracellulare. Il pretrattamento con MSC-EVs ha determinato un recupero parziale della funzionalità, con una frequenza di contrazione aumentata (~15 bpm vs ~5 bpm nel gruppo H₂O₂) e un miglioramento all'efflusso di calcio, simile al gruppo controllo. Conclusioni: Le vescicole extracellulari derivate da MSC hanno mostrato un potenziale effetto cardioprotettivo in un modello in vitro di stress ossidativo, suggerendo un possibile utilizzo nella conservazione dei graft cardiaci. Questi risultati supportano l’ulteriore esplorazione di strategie basate su EVs nel contesto della perfusione ex vivo e della medicina rigenerativa applicata al trapianto di cuore da donatore DCD.
Protective effects of mesenchymal stem cells-derived extracellular vesicles on Cardiomyocytes under oxidative stress.
MAZZUCCO, CHIARA
2024/2025
Abstract
Background: Heart transplantation remains the gold standard treatment for end-stage heart failure. However, the limited availability of donor hearts represents a significant bottleneck. Donation after Circulatory Death (DCD) may expand the donor pool, but it is hindered by prolonged warm ischemia and Ischemia-Reperfusion Injury (IRI), especially in Italy where the functional warm ischemic time is longer than 45 minutes. Since May 2023 60 DCD heart transplant have been performed with a mortality rate of 18%. Innovative strategies for myocardial protection are therefore urgently needed. Aim: This study investigates the protective effects of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in an in vitro model of hypoxia-induced cardiac injury, aiming to explore their potential application in cardiac tissue regeneration and organ preservation for transplantation. Methods: Cardiomyocytes (CMs) were obtained through a 30-day differentiation protocol from human induced pluripotent stem cells (hiPSCs). Morphological and immunohistochemical analyses confirmed the successful differentiation. MSC-EVs were isolated from human umbilical cord-derived mesenchymal stem cells and characterized using Tunable Resistive Pulse Sensing (TRPS) with the Exoid platform. To simulate oxidative injury, hiPSC-derived CMs were exposed to hydrogen peroxide (H₂O₂) for 24 hours. Three experimental groups were analysed via calcium imaging: untreated control CMs, CMs treated with H₂O₂ only, and CMs pre-treated with MSC-EVs before H₂O₂ exposure. Results: Control hiPSC-CMs exhibited synchronized, spontaneous contractions with regular calcium transients and high sarcoplasmic calcium efflux. H₂O₂ treatment significantly impaired contractility, reducing beat frequency and altering calcium kinetics. Pre-treatment with MSC-EVs resulted in partial recovery of physiological calcium dynamics, with increased beat frequency (~15 bpm vs ~5 bpm in H₂O₂-only group) and improved calcium efflux, resembling the control group. Conclusion: MSC-derived EVs showed a promising cardioprotective effect against oxidative stress in vitro, suggesting their potential as therapeutic agents in cardiac graft preservation strategies. These findings support further investigation into EV-based interventions within ex vivo perfusion platforms and cardiac regenerative medicine, particularly in the context of DCD heart transplantation.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/86505