Lung transplantation (LTx) has become the treatment of choice for patients with end-stage respiratory failure and, over the past decades, the worldwide survival of lung transplant patients has increased significantly [1]. Unfortunately, the short- and long-term outcomes of LTxs are still less favorable than other solid organ transplants, the main faults being organ shortage and the fact that more than 80% of potential organ donors are not suitable or used for transplantation [2,3,4,5,6]. Moreover, other factors, such as postoperative graft dysfunction (PGD), infections, and rejection may also contribute to post-transplant mortality [7,8].

Although the use of extended-criteria lung donors and donors after cardio-circulatory death is increasing, there is still wide consensus in the transplant community that a large proportion of potentially viable grafts is discarded due to fear of early graft dysfunction and the increased postoperative morbidity and mortality of the recipient [1,3,9,10]. Ischemia-reperfusion injury (IRI) is the leading cause of early postoperative lung dysfunction, and is characterized by alveolar damage and lung edema usually occurring within 72 hours after LTx [11]. These pathological processes are driven by both oxidative stress and inflammatory pathways, which cause significant damage to the lung parenchyma, leading to the development of early PGD and consequent chronic lung allograft dysfunction [1,10,11,12]. Therefore, a reduction in these adverse effects could significantly improve the success of LTx.

Recently, it has been shown that the use of normothermic ex vivo lung perfusion (EVLP) may be helpful in increasing the number and improving the results of lung transplantation [13,14]. This procedure provides the opportunity to evaluate donor lung function, and different treatments can also be used to further reduce IRI [15,16,17]. Therefore, the improvement of this technique represents a promising strategy to increase the number of suitable organs and to advance LTx.

A suitable approach to improving EVLP technique consists of using mesenchymal stromal/stem cells (MSCs) and/or their secretome, which contains extracellular vesicles (EVs, such as exosomes and EXOs) and other bioactive molecules, including cytokines, chemokines, growth factors, angiogenic and immunomodulatory factors [18,19,20,21]. Although the mechanisms are not fully defined yet, it has been widely demonstrated in both preclinical and clinical studies that therapeutic effects of MSCs are mediated, at least in part, by paracrine factors [21,22,23,24]. MSCs’ therapeutic properties have been also evaluated in a phase II clinical trial to treat acute respiratory distress syndrome (ARDS) [25]. MSCs and/or their products are able to mitigate both lung injury and inflammation in different experimental models, and the infusion of MSCs protects transplanted lungs from IRI [26,27,28,29]. Moreover, it has been found that MSC-based treatment during EVLP is associated with a decrease in ischemic injury of human donor lungs [30,31,32]. In this case, to avoid the founding of infused MSCs in the lung parenchyma, cell-free therapies, including the use of MSC-derived EXOs and MSC-derived conditioned medium (CM), could be considered new approaches to obtaining MSCs’ beneficial effects. EXOs are nanosized structures carrying functional molecules [33], and can be considered a promising therapeutic tool for acute lung injury because they reduce inflammation and enhance tissue regeneration [34,35,36]. Similarly, MSC-derived CM has demonstrated beneficial effects on lung diseases [37,38,39,40,41,42,43]. The use of MSC-derived products rather than direct use of MSCs can prevent all the risks associated with live-cell transplants. Therefore, the use of these products is emerging as a promising approach in the field of LTx.

2. Mesenchymal Stromal/Stem Cell (MSC)-based Therapeutic Approaches to Improve Lung Transplantation

Use of Cells or Their Products	Study Model	Effects Due to MSC Treatment	Mechanisms	References
BM-MSCs	Mouse lung IRI	Protection against cold IRI in lung transplants	Improved arterial blood oxygenation capacity, reduced levels of pro-inflammatory cytokine and cell apoptosis	[29]
MSC-derived EVs	Rat lung IRI and EVLP	Improved tissue integrity and metabolism	Decrease in vascular resistance and rise in perfusate NO metabolites; Up-regulation of genes involved in the resolution of both inflammation and oxidative stress	[34]
UC-MSCs and UC-MSC-derived EVs	Mouse lung IRI	Attenuation of lung dysfunction and injury by improving the efficacy of EVLP	Decreased levels of edema, neutrophil infiltration and myeloperoxidase; decrease in pro-inflammatory cytokines and increase in KGF, PGE2 and IL-10;	[35]
UC-MSC-derived EVs	E. coli-induced rat lung injury	Increased survival	Enhanced phagocytosis of E. coli	[36]
BM-MSCs and BM-MSC-derived CM	Rat lung Injury	Attenuation of lung injury	Reduced levels of pro-inflammatory cytokine	[37]
BM-MSCs and BM-MSC-derived CM	Ventilator-induced rat lung injury	Reduction in injury and improvement in recovery	Reduced levels of edema, neutrophil, and alveolar IL-6 concentrations	[38]
BM-MSC-derived CM	Rat lung IRI	Protection against lung IRI	Decrease in both pro-inflammatory cytokines and infiltrating inflammatory cells, and increase in both M2-like macrophages and regulatory T cells	[39]
AdMSC-derived CM	LPS-induced mouse lung injury	Reduction in ARDS indices	Reduced endothelial barrier hyperpermeability and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.	[40]
AMSC-derived CM	In vitro model of human lung IRI	Attenuation of IRI effects by improving the efficacy of in vitro EVLP	Increase in anti-inflammatory factors and up-regulation of anti-apoptotic factors	[41]
BM-MSCs and AdMSC-derived CM	Rat and human alveolar epithelial cell injury	Decreased cell injury	Decrease in pro-inflammatory factors and increase in anti-inflammatory factors; inhibition of p38 MAPK and translocation of Bcl-2 to the nucleus; Increased expression of cytoprotective glucose-regulated proteins	[43]
MSCs	Swine lung IRI	Attenuation of ischemic injury in donor lungs during EVLP and attenuation of IRI after transplantation	Increased levels of HGF and IL-4 and decreased levels of TNFα and cell death markers	[65]
BM-MSCs	HCL- and LPS-induced rat lung injury	Decreased inflammation	Decrease in proinflammatory cytokines, neutrophil infiltration, hemorrhage and interstitial edema	[122]
UC-MSCs and UC-MSC-derived EVs	Rat neonatal hyperoxic lung injuries	Attenuation of hyperoxic lung injuries	Increased alveolarization and angiogenesis; decrease in alveolar epithelial cell death, macrophages and cytokines in lung	[123]
BM-MSC-derived EVs	Mouse pulmonary arterial hypertension	Reduction in pulmonary vascular remodeling and right ventricle hypertrophy	Increased levels of anti-inflammatory and anti-proliferative miRs including miRs-34a,-122,-124, and -127.	[124]
BM-MSCs	Rat lung IRI	Attenuation of lung pathologic injury	Reduced myeloperoxidase production, decreased levels of of pro-inflammatory cytokine and cell apoptosis in lung tissue	[125]
BM-MSCs	E. coli-induced rat pneumonia	Reduction in lung injury; improvement in survival; reduction in lung bacterial load and suppression of inflammation	Enhanced macrophage phagocytic capacity and increase in lung and systemic concentrations of the antimicrobial peptide LL37	[126]
BM-MSCs	Hyperoxia-induced rat lung injury	Mitigation of emphysema	Increased number of alveoli and decrease in α-SMA expression by myofibroblasts	[127]
BM-MSCs and BM-MSC-derived CM	Cigarette-smoke-induced rat emphysema	Alleviation of emphysema and increase in the number of small pulmonary vessels	Decrease in pulmonary artery medial wall thickness and reduction in apoptosis in lungs with emphysema	[128]
BM-MSCs and BM-MSC-derived CM	LPS-induced mouse lung injury	Resolution of lung injury by attenuating lung inflammation	Decrease in neutrophils and increase in M2 in BAL	[129]
BM-MSCs and BM-MSC-derived CM	Mouse chronic obstructive pulmonary disease	Reduction in injury	Reduced levels of inflammation, fibrosis and apoptotic and increased production of HGF	[130]
AdMSC-derived EVs	Elastase-induced mouse emphysema	Reduction in lung emphysema	Increased levels of FGF2	[131]
BM-MSCs	Bleomycin-induced rat pulmonary fibrosis	Decreased fibrosis	Attenuation of NRF2, NQO1, HO-1, γ-GCS, lipid peroxidation, and increase in SOD activity	[132]
UC-MSCs	Rat lung IRI	Reduction in Oxidative stress damage and inflammation	Reduced levels of MPO activity and neutrophil markers; reduction in reactive oxygen species production	[133]
AdMSCs and AdMSC-derived CM	Sulfur mustard-induced mouse lung injury	Reduction in progressive histopathologic changes in the lung	Reducd levels of both M1 and M2 cells, TNF-α and IL-1β	[134]
BM-MSC-derived CM	Bleomycin-induced rat pulmonary fibrosis	Protection against lung fibrosis	Decrease in lung inflammation, fibrotic scores, collagen deposition, and cell apoptosis	[135]
SHEDs and SHED-derived CM	Bleomycin-induced mouse pulmonary fibrosis	Attenuation of lung injury and improvement in survival rate	Reduced levels of pro-inflammatory factors and increased levels of anti-inflammatory factors and M2 cells	[136]
BM-MSCs	Swine lung transplantation	Improvement in dynamic lung compliance	Reduced intrapulmonary edema	[137]
BM-MSC-derived EVs	E. Coli-induced mouse lung Injury	Reduction in lung edema and inflammation	Decrease in lung protein permeability, neutrophils and macrophage inflammatory protein-2 levels in the BAL fluid; increase in KGF in BAL	[138]
AdMSCs	Rat lung IRI	Attenuation of lung damage after IRI	Suppression of oxidative stress and inflammatory reaction	[139]
UC-MSCs	Swine lung IRI	Attenuation of IRI by improving the efficacy of EVLP	Increased levels of VEGF and decreased concentration of circulating IL-8	[140]
BM-MSCs	Human lung IRI and EVLP	Decreased cold ischemic injury	Decrease in pro-inflammatory cytokines and increase in anti-inflammatory cytokines	[30]
BM-MSCs and BM-MSC-derived CM	E. coli-induced human lung injury	Increase in alveolar fluid clearance in lungs during EVLP	KGF secretion	[31]
BM-MSCs	Human lungs rejected for transplantation and subjected to prolonged ischemic time	Restoration of alveolar fluid clearance	KGF secretion	[32]
BM-MSC-derived EVs	Human lungs rejected for transplantation	Increase in alveolar fluid clearance in donor lungs during EVLP	Improved airway and hemodynamic parameters	[141]
AdMSCs	Clinical trial	Attenuation of IRI and host immunological reaction towards the graft	Not determined	NCT04714801
BM-MSCs	Clinical trial	Attenuation of graft rejection and bronchiolitis obliteran syndrome (BOS)	Not determined	NCT02181712

2.1. Therapeutic Effects of MSCs on Ischemia-Reperfusion Injury (IRI)

Ischemia-reperfusion injury events can occur in the case of stroke or myocardial infarction, as well as in solid organ transplantation. Prolonged ischemia causes deprivation of both oxygen and nutrients, leading to cellular metabolic and ultrastructural pathological changes [142]. Pathological effects mediated by IRI are often potentiated by the onset of inflammation processes, affecting organ quality and transplant outcomes in solid organ transplantation [143]. In this case, IRI-associated injury can be attenuated by cold ischemia storage, but it cannot be completely prevented [144]. Moreover, oxidative stress is a crucial process of IRI-associated effects, leading to the production of toxic molecules [142]. Much scientific evidence shows that MSCs have been able to decrease inflammation and IRI in both in vitro and in vivo models, and these effects are mediated by different mechanisms, including paracrine activity producing a functional secretome [145]. It has been shown that AdMSCs were able to reduce myocardial IRI and to decrease pro-inflammatory cytokine in an in vivo mouse model [146]. In an IRI rat model, Cui et al. demonstrated that AdMSC-derived EXOs were able to protect the myocardium [147].

2.2. MSCs as Therapeutic Tool to Improve EVLP

Focusing on expanding the donor pool to reduce the mortality of lung transplant in recipient patients, different approaches are currently being explored, including the extended criteria for the selection of donors [153], and lung procurement from donors after cardiac death [3]. These strategies have led to the use of the so-called “marginal” organs that do not fulfill the standard criteria of donor lungs. For this scenario, the ex vivo lung perfusion (EVLP) technique was developed in order to: 1. assess graft function after procurement and before implantation; 2. preserve the graft after harvesting; and 3. repair/regenerate potential grafts previously considered unsuitable for transplantation [17,154]. In a lung-transplanted rat model, EVLP treatment was able to protect lung tissue against IRI side effects [155] and, to further reduce IRI complications, pharmacological treatments are possible either by intravascular or endobronchial administration [15,16]. Moreover, although the molecular mechanisms underpinning lung regeneration during EVLP have not been explored yet, recent studies have analyzed metabolic/proteomic events to reveal potential regenerative effects of EVLP [156,157]. This may offer an additional opportunity to evaluate both questionable donor lungs and the effectiveness of new treatments.

The overall data show that the role of MSCs in improving lung recovery appears to be mediated mainly by MSC paracrine signaling; furthermore, the use of MSCs and/or their products, including EVs and CM, represents a promising approach to improving EVLP reconditioning of transplanted lungs. Because of MSCs ability to secrete paracrine factors with anti-inflammatory, anti-oxidant and anti-apoptotic properties, MSCs and/or their products represent a useful tool for integration with EVLP to further preserve/regenerate lung tissue by attenuating IRI during LTx (Figure 1).

Figure 1. Therapeutic strategies to improve ex vivo lung perfusion (EVLP) during lung transplantation. EVLP treatment may sometimes be not sufficient to mitigate ischemia-reperfusion injury (IRI) effects, and lung graft can reveal the lung to have a poor quality after transplantation. The treatment with mesenchymal stromal/stem cells (MSCs) and/or their products (conditioned medium and/or exosomes containing growth factors, cytokines and chemokines) can effectively potentiate EVLP performance and attenuate IRI adverse events by inducing: 1. tissue repair/regeneration; 2. inhibition of reactive oxygen species (ROS); 3. immunomodulation. These effects can lead to the improvement of clinical outcomes after lung transplantation.

3. Conclusions and Perspectives

Many preclinical studies in animal models have clearly shown that the therapeutic effects of MSCs on lung injury, and specifically on ischemia-reperfusion injury after LTx, are mediated by the reduction in inflammation [26,27,28,29,34,35,36,37,39,40,65,125,129,137,140]. Moreover, in a preclinical setting, it has also been demonstrated that MSCs have been able to protect human lungs during transplantation [30,31,32,141]. Interestingly, these effects appear to be related, at least in part, to the paracrine secretion of MSC factors, including proteins and EVs (such as EXOs), which offer an opportunity to develop new cell-free treatments for human lung diseases. On the other hand, only two clinical studies (clinicaltrial.gov, NCT number: NCT04714801 and NCT02181712) are currently testing the potential role of MSC therapy in lung transplantation to avoid graft rejection.

Therefore, although many works have shown the potential application of MSCs in improving the outcomes of lung transplantation, these effects have not been confirmed in appropriate clinical studies yet. Several issues can explain the limited use of MSCs in this field. There are still relevant limitations to the use of MSC therapy in lung transplant clinical practice. MSCs display mutable properties related to the biological and technical variability of their preparation, and these may impact on the therapeutic potency of MSCs [21,88,95]. In particular, the optimal source, dose, and priming of MSCs is crucial and should be optimized. The best EVLP strategy and protocols to treat lungs before transplantation still need to be elucidated. In addition, another important aspect to be clarified is whether the use of MSC-derived products, such as secretome and/or EXOs, can offer additional benefits compared to standard preparations. The understanding of these critical issues could allow the introduction of MSC treatments in clinical lung transplantation. More efficacy studies—for example, in large animals—are needed to determine the best approach for a human clinical setting. Further investigation is needed to better integrate MSC therapy with EVLP use. This new strategy can lead to enhanced EVLP performance, with the ultimate goal of increasing the number of donor lungs and their quality. Moreover, the routine use of EVLP in clinical practice can favor an improvement in lung transplant management and outcomes.