Conditioned Medium from Human Amnion-Derived Mesenchymal Stromal/Stem Cells Attenuating the Effects of Cold Ischemia-Reperfusion Injury in an In Vitro Model Using Human Alveolar Epithelial Cells.

Vitale Miceli,Claudia Carcione,Eva Schmelzer,Mariangela Pampalone,Cinzia Maria Chinnici,Alessandro Bertani,Matteo Bulati,Jörg C Gerlach,Pier Giulio Conaldi,Giandomenico Amico

doi:10.3390/ijms22020510

Abstract

The clinical results of lung transplantation (LTx) are still less favorable than other solid organ transplants in both the early and long term. The fragility of the lungs limits the procurement rate and can favor the occurrence of ischemia-reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) with Steen SolutionTM (SS) aims to address problems, and the implementation of EVLP to alleviate the activation of IRI-mediated processes has been achieved using mesenchymal stromal/stem cell (MSC)-based treatments. In this study, we investigated the paracrine effects of human amnion-derived MSCs (hAMSCs) in an in vitro model of lung IRI that includes cold ischemia and normothermic EVLP. We found that SS enriched by a hAMSC-conditioned medium (hAMSC-CM) preserved the viability and delayed the apoptosis of alveolar epithelial cells (A549) through the downregulation of inflammatory factors and the upregulation of antiapoptotic factors. These effects were more evident using the CM of 3D hAMSC cultures, which contained an increased amount of immunosuppressive and growth factors compared to both 2D cultures and encapsulated-hAMSCs. To conclude, we demonstrated an in vitro model of lung IRI and provided evidence that a hAMSC-CM attenuated IRI effects by improving the efficacy of EVLP, leading to strategies for a potential implementation of this technique.

Highlights

HAMSCs derived from the amniotic membrane of placenta were analyzed by flow cytometry for both positive (CD90, 96.6%; CD73, 98.2%; CD13, 88.30%) and negative (CD45, 0.1%; HLA-DR, 0.1%) surface markers (Figure 2a,b). human amnion-derived MSCs (hAMSCs) were grown in two-dimensional (2D) cultures for 2 passages, and the multipotency of hAMSCs was verified by osteogenic, adipogenic, and chondrogenic differentiation (Figure 2c)
To investigate whether the different culture systems alter the composition of conditioned medium (CM), we used a multiplex-microbead immunoassay to analyze the secretion of different proteins in a CM produced by hAMSCs grown in both 2D and 3D cultures and in a catheter-like device
Our results indicate that the encapsulated-hAMSCs and, even more, the 3D hAMSC-CM, when administered during in vitro Ex vivo lung perfusion (EVLP) have protective effects against Ischemia-reperfusion injury (IRI) induced in lung A549 cells

Summary

Introduction

IRI is driven by several inflammatory pathways (including release of reactive oxygen species, neutrophil activation, and production of proinflammatory factors) that are activated at the time of graft preservation and last until implantation. The activation of such pathways results in a significant damage of the alveolar capillary structures, which can lead to the development of early postoperative graft disfunction (PGD) and, chronic lung allograft dysfunction (CLAD) [4,5]. Hypothermic organ storage is associated with oxidative stress and cell death in alveolar tissue, triggering the activation of proinflammatory pathways after lung transplantation [6]. A reduction of IRI-induced side effects could significantly improve the success of LTx

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Conclusion