MD Alexandre Sitbon
email: alexandre.sitbon@aphp.fr

Pierre-Romain Delmotte

MD Claire Goumard

MD Célia Turco

PhD Jérémie Gautheron

MD Filomena Conti

PhD Lynda Aoudjehane

MD PhD Olivier Scatton

MD PhD Antoine Monsel

Therapeutic potentials of mesenchymal stromal cells-derived extracellular vesicles in liver failure and marginal liver graft rehabilitation: a scoping review

Keywords: Mesenchymal stromal cells-derived extracellular vesicles, Liver transplantation, Acute liver failure, Liver fibrosis, Acute-on-chronic liver failure, Normothermic-machine perfusion

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Acute liver failure (ALF) associates a severe liver dysfunction without pre-existing liver disease. ALF is a rare disease (with an incidence of 10 cases per million per year worldwide) with a heterogeneous presentation, multiple etiology (e.g. acetaminophen (APAP), hepatitis viruses, hepatic ischemia), and in several cases, a lethal outcome [START_REF] Stravitz | Acute liver failure[END_REF] . Pathophysiology of ALF relies on injury induced by specific cause of ALF (e.g., APAP-induced liver injury) and on dysregulated inflammation associated with multiple organ failure [START_REF] Dong | Pathophysiology of Acute Liver Failure[END_REF] . With more than 160 million people suffering from cirrhosis, and approximately 800 000 people dying each year from cirrhosis worldwide, cirrhosis can be considered a major cause of the global burden on health [START_REF] Ye | The burden of liver cirrhosis in mortality: Results from the global burden of disease study[END_REF] . Pathophysiology associates chronic liver inflammation, remodeling of the healthy liver replaced by liver fibrosis and regenerative nods. After an initial asymptomatic phase, cirrhosis evolved from compensated cirrhosis to decompensated cirrhosis (DC). In the case of a precipitating event, DC can evolve towards acute on chronic liver failure (ACLF) [START_REF] Arroyo | The systemic inflammation hypothesis: Towards a new paradigm of acute decompensation and multiorgan failure in cirrhosis[END_REF] combining acute DC with at least 2 organ failures and significant excess mortality within 28 days of hospitalization [START_REF] Arroyo | Acute-on-Chronic Liver Failure[END_REF][START_REF] Moreau | Acute-on-chronic liver failure: A distinct clinical syndrome[END_REF] . ACLF occurs in up to 40% of DC and carries high mortality ranging from 30% to 100% at 1 year, depending on severity [START_REF] Ruiz De Garibay | Critical care hepatology: definitions, incidence, prognosis and role of liver failure in critically ill patients[END_REF] . Sepsis, alcoholic hepatitis, and digestive hemorrhage represent more than 90% of the precipitating event leading to ACLF [START_REF] Trebicka | The PREDICT study uncovers three clinical courses of acutely decompensated cirrhosis that have distinct pathophysiology[END_REF] .

Inflammatory signature in ACLF is characterized by a maladaptive systemic inflammatory response syndrome (SIRS) combining intense systemic inflammation and strong reactive oxygen species (ROS)-induced injury with a close relationship between the intensity of SIRS, the number of organ failures, and mortality on day 28 [START_REF] Clària | Systemic inflammation in decompensated cirrhosis: Characterization and role in acute-on-chronic liver failure[END_REF] . SIRS appears therefore as a major therapeutic target of interest to limit the evolution of ACLF and improve patient survival [START_REF] Arroyo | The systemic inflammation hypothesis: Towards a new paradigm of acute decompensation and multiorgan failure in cirrhosis[END_REF] .

There is currently no specific treatment for ACLF, and only organ support strategies might allow bringing selected patients to liver transplantation (LT), the only curative option for improving the survival of ACLF patients [START_REF] Bajaj | Acute-on-Chronic Liver Failure Clinical Guidelines[END_REF] . However, access to LT remains restricted by a persistent graft shortage and a demographic shift in donor profile (obesity), increasing the incidence of marginal graft. This has led countries to use extended criteria for liver donors (e.g., donation after circulatory death, elderly donors, and high-extended rate of steatosis) inducing an excess risk of ischemia-reperfusion injury (IRI) and primary graft dysfunction, both responsible for altered outcomes [START_REF] Ali | Analysis of ischemia/reperfusion injury in time-zero biopsies predicts liver allograft outcomes[END_REF] , and increased mortality [START_REF] Ito | Ischemia-reperfusion injury and its relationship with early allograft dysfunction in liver transplant patients[END_REF] . Aside from oxidative stress (OS), cell death, and metabolic disruptions, inflammation also plays a crucial role in IRI-induced liver damage by inducing immune activation with innate-adaptive intense immune crosstalk [START_REF] Zhai | Ischaemiareperfusion injury in liver transplantation-from bench to bedside[END_REF] . For those patients, mitigating inflammation and OS might improve liver function and overall survival. Mesenchymal stromal cells (MSCs) are among the innovative immunomodulatory emerging therapies currently being explored. MSCs exert pleiotropic effects including anti-inflammatory, immunomodulatory, anti-fibrotic, pro-metabolomic, anti-apoptotic, and anti-oxidant properties representing a therapeutic option in liver injury [START_REF] Müller | Immunomodulatory Properties of Mesenchymal Stromal Cells: An Update[END_REF] . Although a recent meta-analysis summarizing clinical studies using MSCs in liver diseases suggested that they were safe to use in this clinical setting [START_REF] Thompson | Cell therapy with intravascular administration of mesenchymal stromal cells continues to appear safe: An updated systematic review and meta-analysis[END_REF] , pitfalls remain concerning the use of cell-based therapy (e.g. uncontrolled differentiation or vascular occlusion), which limits its development and clinical use [START_REF] Kholodenko | Mesenchymal Stem Cells in the Adult Human Liver: Hype or Hope? Cells[END_REF] . Thus, MSC-derived extracellular vesicles (MSC-EVs) are opening up new opportunities [START_REF] Silva | Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs -EVOLVE France[END_REF] as these entities naturally tend to mirror their parental cell properties in liver injury while circumventing the pitfalls inherent to the use of MSC. In vitro and in vivo studies demonstrated that MSC-EVs recapitulate the beneficial effects of MSC in reducing cell death and OS, improving hepatocyte function by enhancing autophagy, and mitigating cytokine storm [START_REF] Börger | Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles and Their Potential as Novel Immunomodulatory Therapeutic Agents[END_REF] . This scoping review will describe the potential therapeutic effects of MSCs and MSC-EVs in ALF, liver fibrosis, ACLF, and rehabilitation of marginal liver grafts.

Material and methods

Eligibility criteria

We performed a scoping review by selecting the most relevant articles by combining "Mesenchymal stromal cells", "extracellular vesicles", "exosomes", "acute liver failure", "liver fibrosis", "acute-on-chronic liver failure", "liver transplantation", "liver graft rehabilitation". We have not taken any restrictions regarding the date or location of the publications. The final search occurred on December 12, 2022. We reviewed our findings to identify work relevant to MSC-EVs in liver injury.

Information sources

Our research is conducted in several databases such as PubMed, Web of Science, Cross Ref, and Google scholar.

Mesenchymal stromal cells in liver injury

General properties of mesenchymal stromal cells

MSCs are spindle-shaped cell adherent to plastic, showing multipotent differentiation capacities in vitro, and are characterized by a strong immunomodulation capacity towards key immune cells driving the pathogenesis of SIRS [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] . Main source tissues of MSCs are adipose tissue, bone marrow, placenta, or umbilical-cord tissues [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] . MSCs are well characterized in vitro by the expression of cluster of differentiation (CD) 73, CD90, and CD105, the lack of expression of non-mesenchymal cells (e.g., hematopoietic and endothelial cells) CD11, CD14, CD19, CD34, CD45, CD79, the weak expression of CD106, detectable of human leukocyte antigen-DR isotype. MSCs harbor multi-lineage differentiation potential into osteoblasts, adipocytes, or chondroblasts [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] . MSCs general properties are numerous with among others anti-inflammatory, immunomodulatory, anti-ROS, anti-apoptotic, prometabolomic, anti-fibrogenesis, and pro-regenerative properties [START_REF] Saeedi | A revealing review of mesenchymal stem cells therapy, clinical perspectives and Modification strategies[END_REF] . Immunomodulatory effects of MSCs are mainly paracrine, mediated by the secretion of soluble mediators, organelles, and extracellular vesicles (EVs) into their environment. [START_REF] Müller | Immunomodulatory Properties of Mesenchymal Stromal Cells: An Update[END_REF] Considering their pleiotropic properties (Figure 1), MSCs appear to be a therapeutic candidate of interest for inflammatory diseases and may have several interests for treating liver ALF, LF or ACLF but also in marginal liver grafts rehabilitation.

Mesenchymal stromal cells and acute liver failure

MSCs have been tested in 3 different types of animal models of ALF: surgical (i.e., partial hepatectomy), pharmacological (i.e., APAP, D-galactosamine (D-GalN)), or immune-mediated (i.e., Concavalin A , viral hepatitis) [START_REF] Hefler | Preclinical models of acute liver failure: a comprehensive review[END_REF] . In such preclinical models of ALF, MSCs improved survival, decreased hepatic cytolysis, icterus and inflammation [START_REF] Hu | Improvement of mesenchymal stromal cells and their derivatives for treating acute liver failure[END_REF] . MSCs exert antioxidant properties by enhancing super oxidase dismutase and antioxidant response [START_REF] Cho | Mesenchymal stem cells restore CCl4induced liver injury by an antioxidative process[END_REF] . Altogether, MSCs showed huge potential to alleviate liver injury and improved the regeneration of liver tissues in ALF.

Mesenchymal stromal cells and liver fibrosis

MSCs have been evaluated in following LF models: i) surgical model (e.g., bile duct ligature); ii) chemical model (e.g., Carbon tetrachloride (CCL 4 ), thioacetamide); iii) immune-mediated damage (e.g., Concavalin A); iv) alcohol-induced liver damage; v) diet-induced nonalcoholic steatohepatitis; vi) transgenic model; vii) and organoid liver fibrosis [START_REF] Bao Y Long | Animal and Organoid Models of Liver Fibrosis[END_REF] . In such preclinical models of cirrhosis, MSCs attenuated the progression of fibrosis due to their immunemodulatory properties and could safely improve liver function [START_REF] Zhang | In vivo hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells: Therapeutic effect on liver fibrosis/cirrhosis[END_REF] .

Mesenchymal stromal cells and acute-on-chronic liver failure

Preclinical ACLF models typically include the first hit of chronic injury followed by a second hit of acute liver injury. For example, some authors described the ACLF model using CCl 4induced fibrosis followed by multiple injections of lipopolysaccharide (LPS)/D-GalN to trigger acute liver injury or even the combination of bile duct ligature for fibrosis with LPS to trigger acute liver injury [START_REF] Gama | Animal models applied to acute-on-chronic liver failure: Are new models required to understand the human condition?[END_REF] . In serum porcine, LPS/D-GalN mice ACLF models, intravenous administration of human umbilical cord-derived MSCs (UCMSCs) promoted liver repair, fibrosis resolution, and improved liver function [START_REF] He | Human umbilical cord-derived mesenchymal stem cells improve the function of liver in rats with acute-on-chronic liver failure via downregulating Notch and Stat1/Stat3 signaling[END_REF] . In a similar ACLF model, Liao et al. showed a higher 48-hour survival rate in the UCMSCs-treated group associated with reduced liver cytolysis, and improved liver histology [START_REF] Liao | UMSCs Attenuate LPS/D-GalNinduced Acute Liver Failure in Mice by Down-regulating the MyD88/NF-κB Pathway[END_REF] .

Mesenchymal stromal cells and marginal liver grafts rehabilitation

Facing liver graft shortage and an increase in marginal liver grafts, researchers proposed several approaches to rehabilitate marginal grafts to improve patients' prognosis before and after LT. Recent studies based on animal models highlighted the potential role of normothermic machine perfusion (NMP) in shortening cold ischemia time, increasing organ preservation time, and optimizing organ rehabilitation and regeneration [START_REF] Banker | A Review of Machine Perfusion Strategies in Liver Transplantation[END_REF] . Among therapies tested during NMP, MSCs showed pleiotropic properties to attenuate most of the liver graft IRI in which inflammation plays a key role [START_REF] Owen | Mesenchymal Stromal Cells, a New Player in Reducing Complications From Liver Transplantation? Front Immunol[END_REF] . Recent studies explored the applications of MSCs during NMP, and four of them focused on liver rehabilitation during NMP (rat liver [START_REF] Sasajima | Cytoprotective Effects of Mesenchymal Stem Cells During Liver Transplantation from Donors After Cardiac Death in Rats[END_REF][START_REF] Cao | HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats[END_REF][START_REF] Yang | Bone marrow mesenchymal stem cells combine with normothermic machine perfusion to improve rat donor liver quality-the important role of hepatic microcirculation in donation after circulatory death[END_REF] or porcine liver [START_REF] Verstegen | First Report on Ex Vivo Delivery of Paracrine Active Human Mesenchymal Stromal Cells to Liver Grafts During Machine Perfusion[END_REF] ). The administration of MSCs during NMP improved bile production, decreased hepatic cytolysis, improved liver regeneration, attenuated the level of mitochondrial damage, and enhanced the protective effect of NMP [START_REF] Lascaris | Normothermic liver machine perfusion as a dynamic platform for regenerative purposes: What does the future have in store for us?[END_REF] . Further evidence demonstrated the superiority of NMP plus MSCs to improve the liver graft quality, especially marginal liver grafts [START_REF] Cao | HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats[END_REF] . Figure 2 highlights the therapeutic capabilities proposed by NMP combined with MSCs.

Clinical evaluation of mesenchymal stromal cells in human liver diseases

In ALF, clinical trials showed that MSCs administration was possible and safe. However, despite encouraging results, interpretation remains cautious due to the lack of patients and the relative heterogeneity in their design [START_REF] Shokravi | Mesenchymal stromal cells (MSCs) and their exosome in acute liver failure (ALF): a comprehensive review[END_REF] . In LF, more than ten clinical studies evaluated the impact of MSC-based therapies in liver fibrosis with good safety [START_REF] Mohamadnejad | Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis[END_REF] . Infusion of bone marrow-derived mesenchymal stromal cells (BMSCs) in 9 cirrhotic patients in a randomized placebo-controlled phase I-II single-center study found a significant improvement in the model for end-stage liver disease (MELD) score [START_REF] Schacher | Bone Marrow Mesenchymal Stem Cells in Acute-on-Chronic Liver Failure Grades 2 and 3: A Phase I-II Randomized Clinical Trial[END_REF] . In two studies assessing the impact of BMSCs on alcoholic liver cirrhosis, results showed a good safety profile, an improvement in liver function, and a clear amelioration in liver fibrosis [START_REF] Jang | Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: a pilot study[END_REF][START_REF] Suk | Transplantation with autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: Phase 2 trial[END_REF] . UCMSCs improved liver function documented with enhanced serum albumin levels and reduced total bilirubin level [START_REF] Zhang | Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients[END_REF] . In ACLF, a phase I/II trial evaluated the safety and efficacy of human UCMSCs involving 43 ACLF patients with hepatitis B infection. No significant side effects were observed in UCMSCs group, and UCMSCs increased survival rate, alleviated liver damage, and reduced MELD score [START_REF] Shi | Human Mesenchymal Stem Cell Transfusion Is Safe and Improves Liver Function in Acute-on-Chronic Liver Failure Patients[END_REF] . In another trial, BMSCs infusion was safe in patients with ACLF (n=4) [START_REF] Moreau | Acute-on-Chronic Liver Failure Is a Distinct Syndrome That Develops in Patients With Acute Decompensation of Cirrhosis[END_REF] . Lin et al.

showed that mortality was higher in the control group (n=54) than in the BMSCs group (n=56) in hepatitis B virus-related ACLF (37.0% versus 17.9%, p=0.02) [START_REF] Lin B Liang | Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial[END_REF] . Moreover, a recent meta-analysis of 12 clinical studies showed a beneficial effect of MSCs on liver function in both LF and ACLF (including the MELD score, albumin levels, and coagulation function) [START_REF] Liu | The assessment of mesenchymal stem cells therapy in acute on chronic liver failure and chronic liver disease: a systematic review and meta-analysis of randomized controlled clinical trials[END_REF] .

All these data show that the administration of MSCs in the clinical setting of patients with ALF, cirrhosis, or ACLF is feasible and safe. Several promising signals suggesting beneficial effects are emerging, but the number of well-conducted RCTs is still too limited to conclude that MSCs have a therapeutic effect in these indications.

Actual limitations of Mesenchymal stromal cell-based therapy

Despite a solid rationale, and these promising preclinical and clinical data, the translation and clinical use of MSC-based therapies remain problematic in several ways. First, chromosomic instability and senescence have been demonstrated, and there is a potential tumorigenic effect of MSC-based therapy that has raised concerns [START_REF] Barkholt | Risk of tumorigenicity in mesenchymal stromal cell-based therapies-Bridging scientific observations and regulatory viewpoints[END_REF] . However, no MSCsmediated cancers have been reported in human clinical studies [START_REF] Cao | HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats[END_REF] . Second, MSCs can also be infected by viruses and thus be the vectors of viral dissemination, although this risk has not been shown in clinical studies to date. Third, MSCs are often significantly expanded in vitro to obtain sufficient cells for infusion, which could reduce replicative potential, induce telomere attrition, reduce DNA polymerase and repair efficiency and induce genetic instability which could promote cell senescence and functional alterations [START_REF] Neri | Genetic Stability of Mesenchymal Stromal Cells for Regenerative Medicine Applications: A Fundamental Biosafety Aspect[END_REF] . For clinical trials, cell culture usually lasts to the third or fourth passage to limit genetic instability [START_REF] Martin | MSC Committee of the International Society for Cellular Therapy. A relativity concept in mesenchymal stromal cell manufacturing[END_REF] .

Fourth, although MSCs are immune privileged due to a low immunogenicity profile, they are not protected from the risk of graft rejection and immune-related clearance [START_REF] Zhou | Challenges and advances in clinical applications of mesenchymal stromal cells[END_REF] . Fifth, the manufacturing process of MSCs remains not standardized and there is a myriad of steps introducing heterogeneity like donors characteristics (ages, comorbidity), tissue sources, cell isolation techniques, culture and preservation conditions, which could impact the quality and translational potential of MSC-based therapy [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] . Sixth, pharmacokinetics and pharmacodynamics remain unknown due to MSCs-related factors (e.g., donor age, number of culture passages), and non-MSCs-related factors (e.g., MSCs labeling, host cytotoxic response) [START_REF] Salvadori | Dissecting the Pharmacodynamics and Pharmacokinetics of MSCs to Overcome Limitations in Their Clinical Translation[END_REF] . Seventh, since the manufacturing process is not yet standardized, it is still quite unrealistic to imagine such therapeutics outside of an expert center for cell-based therapies.

Indeed, after expansion of the cells in vitro, it is necessary to cryopreserve the cells (frequently in liquid nitrogen) until needed to maintain their potency by slowing down the metabolic process and then thawing the cells at the patient's bedside before administration [START_REF] Yin | Manufacturing of primed mesenchymal stromal cells for therapy[END_REF] . Eighth, during intravenous injection of MSCs, a large proportion of MSCs are trapped in the lung and could never reach the peripheral organs, notably the liver [START_REF] Zhang | The clinical application of mesenchymal stem cells in liver disease: the current situation and potential future[END_REF] , which explains in part the numerous studies evaluating direct intra-hepatic or intra-splenic injections [START_REF] Sang | Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis[END_REF][START_REF] Amer | Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells[END_REF] .

For those all reasons, special attention has been paid to MSC-EVs as promising therapeutics agents mimicking the pleiotropic effects of MSCs and opening up new opportunities as a cell-free therapeutic approach in liver diseases [START_REF] Silva | Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs -EVOLVE France[END_REF] .

Mesenchymal stromal cells-derived extracellular vesicles in liver injury

Interest in the mesenchymal stromal cells-derived extracellular vesicles

Exosomes were first described in 1983 [START_REF]Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes[END_REF] but were initially considered as cellular waste products. However, in the early 2000s, several studies emerged showing that these nanoparticles harbored a wide variety of bioactive molecules, such as micro ribonucleic acid (miRNA), messenger RNA (mRNA), cytokines, growth factors as well as lipid mediators and other proteins/peptides [START_REF] Tang | Advances in mesenchymal stem cell exosomes: a review[END_REF] . EVs are phospholipid bilayer bound nanoscale vesicles (30-1000 nm with small EVs <200 nm and medium/large EVs >200 nm), released by a wide variety of cell types and include exosomes (30-150 nm), and microvesicles (100-1000 nm). Note that apoptotic bodies (500-5000 nm) and oncosomes (1-10 m) are other types of vesicles, related to EVs but not strictly belonging to the EVs size category [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF] . Together with soluble molecules and organelles, MSC-EVs compose the MSCs secretome. In vitro and in vivo studies demonstrated that MSC-EVs recapitulate the beneficial effects of MSCs in liver disease by reducing cell death and OS and ameliorating hepatocyte function by enhancing autophagy and mitigating cytokine [START_REF] Börger | Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles and Their Potential as Novel Immunomodulatory Therapeutic Agents[END_REF] . MSC-EVs recapitulate the pleiotropic effects of MSCs by transferring their intra-cytoplasmic bioactive cargo (e.g., proteins, lipids, miRNA, mRNA) into target cells [START_REF] Silva | Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs -EVOLVE France[END_REF] with a good immunocompatibility [START_REF] Yoo | Bio-inspired, bioengineered and biomimetic drug delivery carriers[END_REF][START_REF] Sun | Safety evaluation of exosomes derived from human umbilical cord mesenchymal stromal cell[END_REF] . This large body of data shows that MSC-EVs represent a crucial vectoring element of the paracrine therapeutic effect of MSCs. Thus, MSC-EVs could be an innovative therapeutic approach for both immunomodulation and regenerative purposes in liver diseases while circumventing the pitfalls inherent in the use of MSCs [START_REF] Gowen | Mesenchymal Stem Cell-Derived Extracellular Vesicles: Challenges in Clinical Applications[END_REF] .

Compared to MSCs, MSC-EVs have several substantial advantages. First of all, they are not composed of nuclei ("cell-free-based" therapy) leading to a lack of self-replicate capability preventing an excess risk of cancer [START_REF] Barkholt | Risk of tumorigenicity in mesenchymal stromal cell-based therapies-Bridging scientific observations and regulatory viewpoints[END_REF] . Secondly, MSC-EVs are nanovesicles allowing to limit the morphological side effects of MSCs such as pulmonary trapping during intravenous injection with a potential risk of pulmonary embolism and right ventricle overload [START_REF] Coppin | Thrombogenic Risk Induced by Intravascular Mesenchymal Stem Cell Therapy: Current Status and Future Perspectives[END_REF] . Thirdly, MSC-EVs are stable in long-term storage and easily usable in clinical practice, as an off-theshelf medicinal products. Optimizing the lyophilization process of MSC-EVs could contribute to generating long-term stable MSC-EVs formulations at 4°C, and improve MSC-EVs translation into the clinics [START_REF] Trenkenschuh | Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles[END_REF] . MSC-EVs have been demonstrated stable during long-term storage at -80°c up to 6 months [START_REF] Gelibter | The impact of storage on extracellular vesicles: A systematic study[END_REF] and during short-term storage at 0-4°C (up to 8 days) [START_REF] Görgens | Identification of storage conditions stabilizing extracellular vesicles preparations[END_REF] .

MSC-EVs are also more resistant than MSCs to damage caused by freeze-thaw cycles [START_REF] Jeyaram | Preservation and Storage Stability of Extracellular Vesicles for Therapeutic Applications[END_REF] .

Fourthly, MSC-EVs demonstrated a good safety profile in several clinical trials [START_REF] Chinnici | Mesenchymal stromal cell secretome in liver failure: Perspectives on COVID-19 infection treatment[END_REF] . For example, authors showed a good safety and feasibility of the administration of either nebulized or intravenous MSC-EVs in COVID-19 patients [START_REF] Zhu | Nebulized exosomes derived from allogenic adipose tissue mesenchymal stromal cells in patients with severe COVID-19: a pilot study[END_REF][START_REF] Monsel | Treatment of COVID-19-associated ARDS with mesenchymal stromal cells: a multicenter randomized double-blind trial[END_REF] . Finally, MSC-EVs can be modified through bioengineering to be uploaded in specific molecules, carrying specific cargo [START_REF] Park | Enhancement of therapeutic potential of mesenchymal stem cell-derived extracellular vesicles[END_REF] or focusing on selected molecular target [START_REF] Johnson | From Mesenchymal Stromal Cells to Engineered Extracellular Vesicles: A New Therapeutic Paradigm[END_REF] .

We summarized in this review several aspects of MSC-EVs in ALF, liver fibrosis, ACLF, and rehabilitation of marginal liver grafts.

Mesenchymal stromal cells-derived extracellular vesicles and acute liver failure

In animal models of ALF, MSC-EVs improved liver function [START_REF] Damania | Mesenchymal stromal cell-derived exosome-rich fractionated secretome confers a hepatoprotective effect in liver injury[END_REF] with a strong anti-inflammatory effect [START_REF] Zhang | Attenuation of hepatic ischemia-reperfusion injury by adipose stem cell-derived exosome treatment via ERK1/2 and GSK-3β signaling pathways[END_REF] . In this context, MSC-EVs have been shown to reduce hepatic cytolysis [START_REF] Jiang | Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acute liver failure by reducing the activity of the NLRP3 inflammasome in macrophages[END_REF] and limit hepatocyte necrosis [START_REF] Tamura | Immunosuppressive effect of mesenchymal stem cell-derived exosomes on a concanavalin A-induced liver injury model[END_REF] . These effects led to increasing survival in treated rodents in ALF [START_REF] Haga | Extracellular vesicles from bone marrow derived mesenchymal stem cells protect against murine hepatic ischemia-reperfusion injury[END_REF] .

Concerning the underlying mechanisms mediating these effects, authors showed that MSC-EVs reduced apoptosis [START_REF] Nong | Hepatoprotective effect of exosomes from human-induced pluripotent stem cell-derived mesenchymal stromal cells against hepatic ischemia-reperfusion injury in rats[END_REF] by promoting autophagy [START_REF] Zhao | Bone marrow mesenchymal stem cell-derived exosomes attenuate D-GaIN/LPS-induced hepatocyte apoptosis by activating autophagy in vitro[END_REF] and limiting OS-induced liver damage [START_REF] Zhang | Exosomes from adipose-derived mesenchymal stem cells alleviate liver ischaemia reperfusion injury subsequent to hepatectomy in rats by regulating mitochondrial dynamics and biogenesis[END_REF] .

Pro-regenerative effects of MSC-EVs in ALF have also been highlighted [START_REF] Tan | Mesenchymal stem cellderived exosomes promote hepatic regeneration in drug-induced liver injury models[END_REF] . Recent studies highlighted the importance of restoring mitochondrial metabolic function in ALF [START_REF] Zhang | Exosomes from adipose-derived mesenchymal stem cells alleviate liver ischaemia reperfusion injury subsequent to hepatectomy in rats by regulating mitochondrial dynamics and biogenesis[END_REF] . The use of MSC-EVs can inhibit ferroptosis and promote cell regeneration factors [START_REF] Piao | Effects of Exosomes Derived from Adipose-Derived Mesenchymal Stem Cells on Pyroptosis and Regeneration of Injured Liver[END_REF][START_REF] Lin | Mesenchymal stem cells protect against ferroptosis via exosome-mediated stabilization of SLC7A11 in acute liver injury[END_REF] . All studies evaluating the effect of MSC-EVs in liver injury are summarized in table 1.

Mesenchymal stromal cells-derived extracellular vesicles and liver fibrosis

In several preclinical models of liver fibrosis, MSC-EVs have been shown to improve survival [START_REF] Dong | hUCMSC-extracellular vesicles downregulated hepatic stellate cell activation and reduced liver injury in S. japonicuminfected mice[END_REF] , alleviate liver fibrosis, reduce liver inflammation, enhance liver regeneration, induce autophagy, and reduce collagen maturation [START_REF] Bruno | Extracellular Vesicles: A Therapeutic Option for Liver Fibrosis[END_REF] . There is a clear decrease in liver cytolysis [START_REF] Rong | Human bone marrow mesenchymal stem cells-derived exosomes alleviate liver fibrosis through the Wnt/β-catenin pathway[END_REF] , a frequent decrease in proinflammatory cytokines (e.g., Tumor necrosis factor-, IL-6, IL-1, Transforming growth factor-) [START_REF] Ohara | Extracellular Vesicles from Amnion-Derived Mesenchymal Stem Cells Ameliorate Hepatic Inflammation and Fibrosis in Rats[END_REF] , and strong repression in pro-fibrotic genes [START_REF] Mardpour | Extracellular vesicles derived from human embryonic stem cell-MSCs ameliorate cirrhosis in thioacetamide-induced chronic liver injury[END_REF] .

The modulation of innate immunity by blocking the activation of hepatic stellate cells (HSCs)

and by decreasing the quantity of Kupffer cells appear to be a cornerstone of the mechanism of action of MSC-EVs [START_REF] Povero | Human induced pluripotent stem cell-derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis[END_REF] . Authors showed that MSC-EVs inhibited HSCs and induced a transition from the activated to the quiescent form of HSCs through the activation of several signaling pathways (i.e., Wnt/-catenin pathway [START_REF] Rong | Human bone marrow mesenchymal stem cells-derived exosomes alleviate liver fibrosis through the Wnt/β-catenin pathway[END_REF] ). Although the underlying mechanisms remain still not fully understood, one hypothesis expressed by Bruno et al. is that MSC-EVs can alleviate liver fibrosis by restoring a physiological expression of miRNA in the liver downregulating HSCs [START_REF] Bruno | Extracellular Vesicles: A Therapeutic Option for Liver Fibrosis[END_REF] . Table 2 summarized preclinical studies involving MSC-EVs use in liver fibrosis.

Mesenchymal stromal cells-derived extracellular vesicles and acute on chronic liver failure

There is currently only one study focusing on the impact of MSC-EVs in ACLF. Hepatic exosomal miRNAs exploration during MSC-EVs treatment revealed an elevation of several miRNAs (e.g., miR-20a-5p) involved in a strong reduction of inflammation (decreases IL-8 serum levels) [START_REF] Zhang | Potential Networks Regulated by MSCs in Acute-On-Chronic Liver Failure: Exosomal miRNAs and Intracellular Target Genes[END_REF] . Our work has shown that this axis of research is currently underdeveloped and undergoing evaluation. It is a crucial innovative area to develop in the next few years to determine whether it is worth translating into the clinic.

Mesenchymal stromal cells-derived extracellular vesicles and rehabilitation of marginal liver graft

Several teams have successfully performed therapeutic interventions directly on human or animal liver grafts perfused on NMP [START_REF] Buchwald | Therapeutics administered during ex vivo liver machine perfusion: An overview[END_REF] like small interfering RNA (siRNA) uptakes [START_REF] Gillooly | First Report of siRNA Uptake (for RNA Interference) During Ex Vivo Hypothermic and Normothermic Liver Machine Perfusion[END_REF] , defatting cocktails [START_REF] Nagrath | Metabolic Preconditioning of Donor Organs[END_REF] , enkephalin [START_REF] Beal | D-Ala2, D-Leu5] Enkephalin Improves Liver Preservation During Normothermic Ex Vivo Perfusion[END_REF] , or MSCs [START_REF] Yang | Bone marrow mesenchymal stem cells combine with normothermic machine perfusion to improve rat donor liver quality-the important role of hepatic microcirculation in donation after circulatory death[END_REF] , as previously discussed. A recent study by Rigo et al. used human liver stromal cells (HLSC)-derived extracellular vesicles (HLSC-EVs) in an ex vivo NMP rat liver model [START_REF] Rigo | Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model[END_REF] . Findings showed that HLSC-EVs attenuated IRI with a decrease in hepatic cytolysis. They also elegantly demonstrated that MSC-EVs were efficiently internalized into hepatocytes [START_REF] Rigo | Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model[END_REF] . Moreover, human HLSC-EVs-based therapy during NMP in rats liver transplantation from donors after circulatory death reduced liver injury, enhanced liver regeneration, and limited necrosis extension [START_REF] Stefano | Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia[END_REF] . These two studies highlighted the efficiency and feasibility of EVs infusion during NMP procedure, although no study has yet evaluated the impact of human MSC-EVs in an ex vivo perfused large animal or human liver NMP model. We still do not know whether MSC-EVs can rehabilitate marginal grafts, allowing some grafts to be recovered and reconditioned to be eligible for LT and/or to improve the function of other marginal grafts to improve recipients' outcomes [START_REF] Stefano | Stateof-the-Art and Future Directions in Organ Regeneration with Mesenchymal Stem Cells and Derived Products during Dynamic Liver Preservation[END_REF] . This is a field of research that deserves to be explored in the coming years.

MSC-EVs in human studies

Regarding the clinical use of MSC-EVs, a dozen studies have been published and another dozen are in the process of including patients [START_REF] Zhou | Challenges and advances in clinical applications of mesenchymal stromal cells[END_REF] . Findings demonstrate the feasibility and safety of administering clinical-grade MSC-EVs to patients. None of these studies have focused on liver disease. Whether in ALF, cirrhosis, or ACLF, no clinical study testing MSC-EVs has yet been conducted to the best of our knowledge. The good safety records of clinical studies using MSCs and the compelling preclinical data concerning MSC-EVs in liver injury led us to expand the investigation of MSC-EVs in liver injury and to consider MSC-EVs as a promising future therapy. This is an innovative avenue of research, with a solid rationale that has yet to be consolidated. Several questions remain to be addressed before we can advance to the first phases of clinical development.

Discussion

When considering the clinical transfer of MSC-EV, several heterogeneities emerge at multiple levels that are discussed here, including the tissue of origin (bone marrow, adipose tissue, human umbilical cord for example), the culture conditions (hypoxia cell, stimulation by growth factors for example), the therapeutic dose, and the optimal mode of administration of MSC-EVs [START_REF] Rohde | Manufacturing and characterization of extracellular vesicles from umbilical cord-derived mesenchymal stromal cells for clinical testing[END_REF] .

Manufacturing challenge. MSCs need to be expanded multiple times to generate enough cells to reach therapeutic doses of MSC-EVs. Scaling-up cell production involves costly large batch culture devices limiting its availability. Several bioreactors (e.g., three or twodimensional bioreactors) are currently developed to allow large scale manufacturing of MSC-EVs [START_REF] Johnson | From Mesenchymal Stromal Cells to Engineered Extracellular Vesicles: A New Therapeutic Paradigm[END_REF] .

MSC-EVs production.

There is also considerable heterogeneity in the techniques used at each stage of MSC-EVs production, and the optimal techniques for expansion (e.g., 2D versus 3D, with or without microcarriers) and MSCs stress (e.g., hypoxia versus serum starvation), extraction, and purification of MSC-EVs (e.g., ultracentrifugation versus tangential flow filtration) have yet to be determined [START_REF] Maumus | Mesenchymal Stem Cell-Derived Extracellular Vesicles: Opportunities and Challenges for Clinical Translation[END_REF] .

Best parental cells.

The selection of the best tissue of origin and parental stromal cells as well as the qualitative and quantitative screening of MSC-EVs remains to be determined and should be standardized. Selection of the donors remains a question of importance due to the potential risk of spreading viruses and the poor quality of MSCs derived from specific patients [START_REF] Shokravi | Mesenchymal stromal cells (MSCs) and their exosome in acute liver failure (ALF): a comprehensive review[END_REF] .

Standardizing characterization. Although the International Society for Cell and gene

Therapy ® recommendations have made progress towards standardization of the techniques used to characterize EVs, there is still some heterogeneity in the combination of characterization panels, including with different levels of priority, nanotracking analyzer counting, protein, and mRNA quantification, electron microscopy imaging, westernbloting, flow cytometry detected marker expression panel [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF] . This heterogeneity induces a potentially important variability in the therapeutic effects of the EVs used, rendering the comparison between studies challenging. The best-standardized potency assays also remain to be established to guarantee a certain level of clinical therapeutic effect.

Route of administration.

While MSC-EVs are rapidly eliminated in the blood with a short plasma half-life 100 , they are still detectable in the liver 24 hours after infusion 101 . Thus, the best administration routes to expect conclusive results in human studies still need to be investigated (i.e., intravenous versus intrasplenic, portal, or intra-arterial injection) [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF] .

Standardizing the therapeutic dose. To achieve a similar therapeutic effect, the dose of MSC-EVs used in published studies is commonly higher than the equivalent dose of MSCs 102 . However, there is a broad range of therapeutic doses used in animal studies, spanning 0.1 to 250 g of protein or 2.0x10 8 to 5.0x10 11 particles per dose, assuming that 1 g of protein equates to around 2.0x10 9 MSC-EVs 103 . Optimal treatment regimen (single versus multiple injections) remains also to be defined.

Bioengineering of MSC-EVs. Further studies explored the impact of either EVs released from upstream pre-treated MSCs or downstream MSC-EVs modified after MSCs release. Indeed, due to their ability to carry many active molecules, MSCs-EVs can be exploited as drug delivery systems and can be modified (e.g., uploaded) to deliver specific therapeutic agents.

MSCs-EVs can be viewed as cargo vectors with high stability in the bloodstream, with specific targeting capabilities, and the ability to cross physiological barriers by pre-treatment and/or gene modification 104 . Studies using modified MSC-EVs in ALF, LF and, ACLF are summarized in the Table 3.

Conclusion and perspectives

Finally, EVs derived from several MSCs sources have confirmed beneficial properties such as anti-inflammatory, immunomodulatory, pro-regenerative, anti-apoptotic, antioxidant, antifibrotic, and pro-metabolomic effects in rodent models of ALF, ACLF, and LF. While circumventing critical pitfalls inherent to their parent cells, MSC-EVs offer a cell-free-based therapeutic approach for liver diseases by recapitulating the effects of MSCs. Evidence regarding the effects of MSC-EV in ACLF and marginal liver graft rehabilitation remains to be expanded. These are areas that have not yet been fully explored. Although good safety records of clinical trials testing MSC-EVs in other contexts than liver diseases have been published, the clinical impact of MSC-EVs on liver disease remains unknown and needs to be investigated. Some questions still need to be addressed such as the optimal tissue of origin of MSCs, MSC-EVs characterization, route of administration, therapeutic dose, cost of production, and mechanisms of action. The prospect of using bioengineered MSC-EVs as a carrier for targeted therapy is also attractive. Finally, there is still a long way before using MSC-EVs in daily life at the bedside and to be able to define a clear framework for using MSC-EVs in liver injury.

" Take-home message":

Mesenchymal stromal cells-derived extracellular vesicles are a future innovative approach in acute liver failure, cirrhosis, acute-on-chronic liver failure, and rehabilitation of marginal liver graft due to their pleiotropic actions (e.g., anti-inflammatory, immunomodulatory, proregenerative, anti-apoptotic, antioxidant, anti-fibrotic, pro-metabolomic effects). Expanding preclinical findings and developing clinically relevant models are needed to confirm promising emerging results, and before advancing one step further their clinical translation into first clinical phases trials.

Figure 3 ,

 3 summarizes the potential impact of MSC-EVs on liver injury

Figure 1 :

 1 Figure 1: Classical sources of mesenchymal stromal cells, general properties and perspective in liver diseases

Figure 2 :Figure 3 :

 23 Figure 2: Overview of exosome of mesenchymal stromal cells expected effects in liver diseases Figure 3: Perspectives in graft rehabilitation

Table Legends Table 1 :

 Legends1 Preclinical data of mesenchymal stromal cells-derived extracellular vesicles on acute liver failure, acute-on-chronic liver failure and ischemia-reperfusion liver injury.

Table 2 :

 2 Preclinical data of mesenchymal stromal cells-derived extracellular vesicles on liver fibrosis.

Table 3 :

 3 Bioengineering applications of mesenchymal stromal cells-derived extracellular vesicles in liver injury.

Availability of data and material:

The data that support the findings of this study are available from the corresponding author, AS, upon reasonable request.

Abbreviations

EVs

Extracellular vesicles

Declarations

Funding: there is no funding related to this study.

Conflicts of interest/Competing interests:

The remaining authors declare having nothing to disclose.

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