Ischemia-reperfusion injury (IRI) represents a major challenge in liver transplantation, driving acute dysfunction and contributing to long-term allograft rejection. This process triggers a robust inflammatory response, leading to hepatocyte damage, senescence, and impaired liver regeneration. While the underlying mechanisms remain incompletely understood, increasing evidence highlights macrophage-derived signaling as a pivotal driver of hepatocyte fate during IRI. Here, we identify iRhom2 as a key regulator of immune-mediated liver injury, orchestrating macrophage-driven inflammation and hepatocyte senescence. iRhom2 is known to modulate the secretion of multiple cytokines by macrophages, yet its specific contribution to IRI-driven hepatocyte senescence has not been fully elucidated. We reveal a significant upregulation of iRhom2 in IRI+ reperfused allografts, particularly in Kupffer cells and monocyte-derived macrophages. Functional characterization in iRhom2-deficient macrophages revealed reduced ER stress, preserved mitochondrial function, and attenuated apoptosis, indicating a protective role against IRI-induced cellular damage. Proteomic profiling further uncovers iRhom2-dependent secretion of inflammatory mediators, with HMGB1 emerging as a critical damage-associated molecular pattern (DAMP) molecule in this context. Notably, HMGB1 release occurs independently of TACE catalytic activity, suggesting an alternative unexplored regulatory mechanism. Furthermore, co-culture experiments confirm that macrophage-derived HMGB1 directly induces senescence of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) under in vitro IRI condition, driving the up-regulation of key senescence markers and disrupting cell cycle dynamics. Strikingly, HMGB1 neutralization enhances hepatocyte viability and mitigates senescence, underscoring its pathogenic role. Additionally, HMGB1 knockdown in macrophages protects hepatocytes, though p21 expression remains unaffected, hinting at additional senescence pathways. Our findings establish iRhom2 as a central orchestrator of macrophage-driven hepatocyte dysfunction in IRI and suggest that targeting the iRhom2-HMGB1 axis could represent a promising therapeutic strategy to improve post-transplant liver recovery and long-term graft survival.

Age-related joint diseases are driven by structural deterioration, chronic inflammation, and impaired regenerative capacity. Mesenchymal stromal/stem cells (MSCs) have emerged as promising therapeutic candidates due to their immunomodulatory and regenerative effects, largely mediated by their secretome and extracellular vesicles (EVs). However, the therapeutic efficacy of MSC-derived products depends on both the cell source and the conditioning stimuli to which MSCs are exposed. In this study, we characterized secretomes derived from placenta-derived MSCs (PDSCs) and adipose-derived MSCs (ASCs) by performing a comparative proteomic and exosomal microRNA (miRNA) profiling. Moreover, the impact of different priming strategies, including hypoxia, interferon-gamma (IFNγ), and interleukin-1 beta (IL1β), on the paracrine properties of these cells was assessed. Proteomic analysis identified over 7,000 proteins, with PDSC secretome enriched in pathways related to osteogenesis, chondrogenesis, extracellular matrix organization, angiogenesis, and immune regulation, whereas ASC secretome displayed limited enrichment in these processes. Functional scoring highlighted IL1β priming as the most effective strategy to enhance osteochondral and immunomodulatory protein signatures in PDSCs. Differently, IFNγ priming selectively expanded the repertoire of exosomal miRNAs, with enrichment in signalling networks including Wnt/β-catenin, TGF-β, NF-κB, and T cell receptor pathways, underscoring its role in fine-tuning immune and regenerative functions. Together, our findings revealed that PDSCs secrete a broader and more functionally relevant spectrum of bioactive molecules for osteochondral applications compared with ASCs. Moreover, distinct priming strategies showed to differentially regulate cell paracrine outputs. In particular, while IL1β primarily enhances protein-driven regenerative and immunomodulatory activity, IFNγ promotes a functionally enriched exosomal miRNA cargo. These results indicate PDSC-derived secretomes as versatile candidates for next-generation, cell-free therapies in joint diseases.

BackgroundThe clinical management of bloodstream infections (BSIs) caused by KPC-Klebsiella pneumoniae has been severely challenged by the emergence of ceftazidime/avibactam (CAZ/AVI) resistance. The circulation of CAZ/AVI-resistant KPC-Kp remains a public health critical concern, warranting the need of strict epidemiological surveillance and genomic analysis to elucidate the underlying molecular mechanisms.MethodsWe retrospectively investigated 16 BSIs caused by KPC-Kp isolates resistant to CAZ/AVI collected from January 2022 to July 2025. Antimicrobial susceptibility testing (AST) was performed to assess resistance profiles against last-line agents. Whole Genome Sequencing (WGS) and bioinformatic analyses were employed to identify molecular drivers of resistance and evaluate genetic relatedness. Patients’ data were collected to integrate clinical outcomes with the microbiological features of the isolated strains.ResultsThe BSIs analyzed were characterized by a high degree of severity, with 9/16 patients presenting a Pitt bacteremia score ≥ 4 and 7/16 (44%) a 30-days mortality rate. Intestinal colonization by KPC-Kp was identified in 14/16 patients, and prior CAZ/AVI-based therapy had been administered to 12/16, suggesting the resistance development under selective antimicrobial pressure. AST revealed resistance to last-line agents: imipenem/relebactam (4/16), meropenem/vaborbactam (2/16) and colistin (3/16), alongside reduced susceptibility to cefiderocol (11/16), thereby limiting effective treatment strategies. Besides the predominance of high-risk clones, such as ST101 (8/16) and ST512 (3/16), phylogenetic analysis highlighted a cluster within ST101 group, suggesting a hospital outbreak. Furthermore, two novel sequence types, ST9507 and ST8640 were identified. Multiple KPC variants were detected, including the novel KPC-293 and KPC-296, which were not previously described. All isolates exhibited a truncated OmpK35 in combination or not with a highly mutated OmpK36, with insertion in loop3 being the most prevalent alteration. Given the heterogeneity of the KPC variants found in the isolates, and the consistent loss of porin function, the OmpK35/36 impairment may represent the pivotal driver of the MDR phenotype in these strains.ConclusionOur findings indicate that the synergy between multiple KPC variants and porin structural alterations may represent a critical determinant underlying the clinical challenge posed by CAZ/AVI resistance in invasive infections.

BACKGROUND:Safety and immunological activity of donor blood monocyte-derived regulatory dendritic cells (DCreg) generated ex vivo have been demonstrated recently in living donor liver transplantation. A potential alternative/additional source of DCreg precursors is the allograft perfusion fluid. Our goal was to establish the extent to which DCreg could be generated from CD14 + myeloid precursors in living donor liver perfusate (LD-LP) and to assess the phenotype and immunoregulatory properties of the perfusate-derived DCreg (LP-DCreg). METHODS:Presumptive DCreg were generated from CD14 + precursors recovered from n = 14 right lobe liver grafts using GM-CSF + Interleukin (IL)-4, with the addition of vitamin D3 and IL-10. Control immature DC and corresponding Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA)-stimulated DC populations were also generated. Cell phenotype and function, DCreg maturation resistance, T-cell allostimulatory activity, and the roles of immunoregulatory molecules in llograft perfusate (LP)-DCreg function were analyzed by flow cytometry, RT-PCR analysis, cytokine assays, and MLR. RESULTS:Presumptive DCreg generated from live donor-LP comprised relatively high proportions of CD141 hi CD163 + cells. They expressed comparatively high coinhibitory programmed death ligand-1 (PD-L1):costimulatory molecule (CD86) ratios, even after MPLA stimulation. Moreover, unlike immature DC, they produced high levels of IL-10 but minimal/low proinflammatory IL-12 and tumor necrosis factor alpha following MPLA stimulation. These LP-DCreg were poor stimulators of naïve allogeneic T cells and suppressed alloreactive CD4 and CD8 T-cell proliferation. Blocking of the PD-L1-PD-1 signaling pathway reversed their regulatory function. CONCLUSIONS:Liver graft perfusate constitutes a novel source of myeloid precursors for DCreg generation.

Introduction:Liver regeneration is essential for successful outcomes after liver transplantation. However, ischemia-reperfusion injury (IRI) remains a major determinant of graft dysfunction that can profoundly affect hepatic regenerative responses. Although ischemic stress has been implicated in both tissue damage and regenerative signaling, its direct impact on progenitor differentiation and hepatocyte maturation remains poorly understood, partly due to the lack of controllable human experimental models. In this study, we investigated how controlled ischemic stress influences hepatocyte differentiation and regenerative signaling using a human liver organoid (HLiO) model. Methods:Organoids were expanded as undifferentiated cultures and subjected to a stepwise differentiation protocol toward hepatocyte-like cells. An in vitro IRI model was generated by exposing organoids to 16 h of cold ischemia (O2 0%) followed by reperfusion under normoxic conditions (O2 20%). Molecular, imaging, and functional analyses were performed to evaluate progenitor status, hepatocyte differentiation, and the release of inflammatory mediators. Results:Differentiation of HLiOs induced a shift from progenitor-associated gene expression toward hepatocyte-specific programs, accompanied by increased albumin secretion and expression of mature markers. Controlled ischemia caused a transient reduction in viability and triggered the release of High Mobility Group Box 1 (HMGB1), Interleukin 1 Beta (IL-1β), Interleukin 8 (IL-8), and Oxidized Low Density Lipoprotein Receptor 1 (LOX-1), followed by recovery during reperfusion. Notably, ischemic preconditioning enhanced hepatocyte maturation, characterized by stronger downregulation of progenitor markers, increased expression of Cytochrome P450 3A4 (CYP3A4), Hepatocyte Nuclear Factor 4 Alpha (HNF4A), Alpha-1 Antitrypsin (A1AT), and albumin, and improved functional output compared with standard differentiation. Discussion:These findings suggest that sub-lethal ischemic stress may act as a regenerative stimulus, potentially mediated by a progenitor-associated HMGB1-LOX-1-IL-8 signaling axis. Despite the absence of non-parenchymal liver cells, this organoid platform provides a controllable system to study intrinsic regenerative responses to ischemia, and indicates that appropriately modulated ischemic cues might promote hepatocyte differentiation, and improve graft recovery after liver transplantation.

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic approach for the treatment of oncological malignancies such as hepatocellular carcinoma (HCC). In this work, we have engineered primary human NK cells, re-directing them so they can specifically recognize Glypican-3 (GPC3), an immunotherapeutic target for HCC. In previous studies, we have demonstrated that IFN-α significantly enhances NK cells' anti-tumor and anti-viral cytotoxicity. Fourth-generation self-inactivating lentiviral vectors were used to deliver a transgenic expression of IFN-α or its co-expression with IL-15 (which induces NK cells expansion, survival, and function), aiming to enhance CAR-GPC3 NK cells' anti-tumor response against HCC. We optimized a protocol for efficient transduction of primary NK cells, demonstrating that CAR expression is maintained at high levels over time. Exposure of HCC ectopically expressing GPC3+ to CAR-GPC3-IL15 and CAR-GPC3-IL15-IFNα NK cells demonstrated significant in vitro cytotoxicity and cytokine production, dependent on GPC3 expression. To prevent undesired side effects of CAR-NK cell immunotherapy, co-delivery with a suicide gene is advised as a safety measure. Thus, a truncated epidermal growth factor receptor (tEGFR) was co-delivered with the anti-GPC3 CAR, which efficiently promoted the suicide of the CAR-NK used in this work. Our study demonstrates the efficacy of re-directed CAR-GPC3 primary NK cells, encouraging further preclinical and clinical translation studies and strengthening the potential of these cells as a novel treatment option for patients with HCC.

The therapeutic potential of extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) is limited by the lack of standardized, Good Manufacturing Practice (GMP)-compliant production protocols. This study investigates the effects of MSC-Brew, a commercially available GMP-grade medium, on MSC-derived EVs in comparison to those produced in conventional cultures with DMEM supplemented with 10% fetal bovine serum (FBS). MSCs from adult dermis were successfully isolated and expanded in Brew medium while retaining their characteristic surface marker expression. MSC-EVs derived from Brew cultures met the Minimal Information for Studies of Extracellular Vesicles (MISEV) criteria, including particle size, concentration, marker expression, and minimal inflammatory cytokine content. Notably, Brew-EVs exhibited a significantly higher particle-to-protein ratio compared to EVs produced in FBS-containing cultures, indicating improved purity. Proteomic analysis revealed a largely conserved composition between Brew-EVs and conventionally produced EVs, and microRNA (miRNA) profiling identified only four differentially expressed miRNAs. Brew-EVs were enriched in anti-fibrotic miRNAs and effectively reduced collagen secretion in transforming growth factor (TGF)-β1-activated LX-2 cells, a human hepatic stellate cell line used as a model of liver fibrosis. These findings support MSC-Brew medium as a standardized, serum-free platform for the consistent production of high-quality EVs suitable for therapeutic applications.

Human pegivirus (HPgV) is a positive-sense, single-strand RNA virus belonging to the Flaviviridae family. Although not conclusively linked to a specific disease, an increasing number of studies have recently reported an association between this virus and different human pathologies. In this study, we present a 6-month-old female infant admitted to the hospital for severe acute hepatitis. Her clinical history started with a one week of fever and diarrhea treated with paracetamol and amoxicillin–clavulanate for a total of 4 days. The persistence of the symptoms, high levels of transaminases, coagulopathy, increased lymphocytosis, and C-reactive protein (CRP) in the blood suggested an acute hepatitis episode. Serological and molecular biology tests for hepatotropic and non-hepatotropic viruses, including hepatitis B virus (HBV), hepatitis A virus (HAV), hepatitis C virus (HCV), hepatitis E virus (HEV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV), enterovirus, and adenovirus, were negative. Metabolic and genetic alterations, deficiency of alpha-1 antitrypsin, and Wilson’s disease were ruled out following negative results. The child was thus treated with supportive therapy. Metagenomic next-generation sequencing (mNGS) performed to identify other possible infective agents undetected with the classical tests, showed the presence of the complete genome of human HPgV-1. This case provides further evidence supporting the hypothesis of the pathogenic role of HPgV-1 and warrants particular attention, especially in the pediatric population. Moreover, here we confirmed the diagnostic power of metagenomic-NGS in the detection of unusual pathogens.

Lung transplantation (LTx) significantly improves outcomes for patients with end-stage respiratory failure. However, primary graft dysfunction (PGD) remains one of the most relevant hurdles. Although PGD is attributed to ischemia-reperfusion injury (IRI), immune responses, primarily T cell-mediated, may play a pivotal role in its pathogenesis. Additionally, innate immune activation following IRI links PGD to adaptive alloimmunity, highlighting the impact of early events on LTx outcomes. Immune checkpoints (ICPs) such as PD-1/PD-L1, CD40/CD40LG, and OX40/OX40L, regulate post-LTx T cell responses, and dysregulation of microRNAs (miRNAs) has been implicated in altering ICP expression, influencing the amplification of immune responses.In this preliminary study, we used the taqMan low-density array (TLDA) cards to investigate miRNA dysregulation’s prognostic potential as a PGD marker in pre-transplant back-table lung biopsies. Our analysis revealed differential miRNA expression in donor lung tissues, potentially associated with PGD onset, targeting immune regulatory pathways. Specifically, deregulated miRNAs targeted key ICP proteins, including PD-L1, CD40LG, and OX40L. Moreover, the differential expression of these miRNAs was observed in grafts with future PGD compared to grafts without PGD, suggesting a potential prognostic benefit and a possible role for lung tissue miRNAs in the onset of early graft dysfunction.These findings provide a basis for future investigations into their mechanistic roles and therapeutic potential for PGD. Although based on a limited number of cases, our results imply that miRNAs might be involved in early graft dysfunction. While requiring validation in larger cohorts, our data raise the possibility that the evaluation of the aforementioned markers during the pre-transplant phase, might offer a prognostic benefit in monitoring the onset of PGD. Additionally, the use of compounds that can modulate the function of these molecules could be evaluated for the management of LTx patients.

Background/Objectives: Type 1 diabetes (T1D) is a chronic autoimmune condition characterized by the destruction of pancreatic β-cells, leading to insulin deficiency. Current therapies, such as islet transplantation, face significant challenges, including limited donor availability and the need for lifelong immunosuppression. Encapsulation technologies offer a promising alternative, providing immune protection and maintaining β-cell viability. In this study, we propose an encapsulation device featuring a spiral tubular semipermeable polyethersulfone (PES) membrane reinforced with a rigid biocompatible resin scaffold. Methods: The PES membrane was engineered with a tailored porosity of 0.5 µm, enabling efficient nutrient and oxygen exchange while preventing immune cell infiltration. Using INS-1E insulin-secreting cells aggregated into size-controlled islet-like spheroids (ILSs), we evaluated the device’s performance. Results: The device achieved high ILS viability and insulin secretion over 48 h at therapeutic densities, maintaining functionality comparable to free-floating ILSs (control). The PES membrane, with its mechanical stability and biocompatibility, ensured durability without compromising diffusion dynamics, overcoming a critical limitation of other encapsulation approaches. Importantly, the device geometry allowed for the encapsulation of up to 356,000 islet equivalents (IEQs) in a single capillary fiber, reaching therapeutic thresholds for T1D patients. Conclusions: this device, with its innovative design, enables high-density encapsulation while preserving ILS functionality and scalability, making it a potential platform for clinical application. This work highlights the potential of PES-based encapsulation devices to overcome key barriers in T1D treatment, paving the way for personalized, long-term solutions to restore insulin independence.

Extracorporeal membrane oxygenation (ECMO) is a vital intervention for patients with severe respiratory failure, particularly in unresponsive acute respiratory distress syndrome (ARDS) cases. However, patient selection for ECMO remains a significant challenge. This study aims to identify novel immune-based biomarkers to improve eligibility assessment and predict outcomes in critically ill COVID-19 patients undergoing ECMO. This monocentric observational retrospective cohort study included 80 patients with severe COVID-19-related pneumonia who required ECMO support due to unresponsive ARDS. The patients were admitted to the intensive care unit (ICU) of IRCCS-ISMETT Hospital between September 2020 and April 2021, before the availability of COVID-19 vaccines. All patients were infected with the original SARS-CoV-2 Wuhan strain. Using machine learning approaches, the study analyzed clinical and laboratory data, cytokine levels, RNA sequencing (RNA-seq), and immune cell profiles collected within two days of hospitalization. The analysis identified a 5.56-fold increased mortality risk in patients presenting with a combination of immune factors: a T cell exhaustion profile, low interferon-alpha (IFNα) levels, and high calprotectin levels. These immune markers were strongly associated with poorer outcomes in patients undergoing ECMO. Our findings highlight the critical role of immune profiling in ECMO patient selection and outcome prediction. Incorporating immune-based biomarkers into clinical assessments may enhance the evaluation of ECMO eligibility and guide treatment decisions, ultimately improving patient outcomes.

OBJECTIVE:In recent decades, the increasing prevalence of multidrug-resistant Klebsiella pneumoniae carbapenemase (KPC)-carrying K. pneumoniae (KPC-Kp) has become a worldwide public concern. Herein, we characterised a ceftazidime/avibactam (CAZ/AVI), meropenem/vaborbactam (MER/VAB) and imipenem/relebactam (IMI/REL)-resistant KPC-Kp strain isolated from a critically ill transplant patient. METHODS:Antimicrobial susceptibility testing and whole-genome sequencing (WGS) were conducted to characterise the strain at phenotypic and genotypic levels. Genomic DNA was sequenced using the Illumina platform. Bioinformatic analyses were used to investigate the genome sequences both for resistance and virulence features, and for the characterisation of plasmids. RESULTS:Phenotypic characterisation revealed that the KPC-Kp isolate was highly resistant to a wide range of antibiotics, including all β-lactam/β-lactamase inhibitor combinations such as CAZ/AVI, MER/VAB, IMI/REL and cefiderocol. WGS analysis showed that the isolate, belonging to the rare lineage ST661, contained several resistance and virulence genes. Among the resistance genes, we identified a new KPC variant within the mobile genetic element Tn4401-KPC-245-characterised by the insertion of nine amino acids (RAPNKDDYT) at position 263 as well as an amino acid change within the protein sequence, E274D, compared with KPC-3. Interestingly, the presence of mutations only in the blaKPC gene and not in other β-lactamase coding genes strongly points to the role of KPC-245 in β-lactam/β-lactamase inhibitor combinations and cefiderocol resistance. CONCLUSIONS:In our study, by using WGS analysis on a clinical isolate, we identified a new blaKPC variant within the Tn4401 transposon. Our results confirm the importance of continuous surveillance of multidrug-resistant K. pneumoniae in the clinical context.

Metallo-β-lactamases (MBLs)-producing Enterobacterales infections represent a serious threat in clinical practice due to high mortality rates associated and the limited therapeutic options available. Given their increased dissemination across Europe and their involvement in hospital outbreaks, further epidemiological investigations are required. Here, we provide a comprehensive characterization, by integrating clinical, genomic, and phenotypic data, of 22 MBL-carrying Enterobacterales strains (17 Klebsiella pneumoniae and 5 Escherichia coli), isolated from critically ill patients admitted to IRCCS ISMETT from 2021 to 2024. Bacterial antimicrobial susceptibility was evaluated using phenotypic methods. Using a whole-genome sequencing approach, we identified the sequence types (STs) and depicted the resistome and virulome content. This study revealed the circulation of alarming MBL-producing strains in our hospital, with New Delhi MBL (82%), followed by Verona integron-encoded MBL (VIM) (18%). Beyond carbapenem resistance, these strains exhibited high resistance rates to crucial antibiotics, including amikacin (63%), in addition to reduced susceptibility to cefiderocol (50%). Moreover, the emergence of VIM-1 K. pneumoniae isolates resistant to colistin was observed. Genomic analysis revealed the presence of high-risk clones such as K. pneumoniae ST147 and ST512 and E. coli ST648, necessitating vigilant monitoring. The resistome analysis showed widespread co-occurrence of several resistance genes, contributing to the multidrug-resistant (MDR) phenotype. The convergence of resistance and virulence characteristics highlights the urgent need for integrated genomic surveillance and improved measures to control the spread of hypervirulent MDR bacteria.IMPORTANCEThe increasing prevalence of metallo-β-lactamase-producing Enterobacterales is a significant healthcare concern. Traditional phenotypic methods, commonly employed in diagnostic labs, are essential for the resistance marker detection and antimicrobial susceptibility testing. Instead, bacterial genetic features, including additional resistance genes and virulence determinants that have significant clinical implications, remain neglected. To overcome this critical gap, an integrated approach, consisting of phenotypic and genomic analyses, is crucial for gaining essential insights that guide both comprehensive patient management and effective epidemiological surveillance. Our findings highlight the importance of antimicrobial stewardship, antimicrobial susceptibility testing, and control measures to limit the spread of high-risk multidrug-resistant bacteria.

Immunosuppressive therapies used in clinics to reduce the risk of rejection in transplanted patients unfortunately also decrease the response of the immune system to the pathogens. Previous data has shown that the most diffuse SARS-CoV-2 variants of concern between 2020 and 2021 showed a different modulation of the host immune response in healthy subjects, with the Delta B.1.617.2 variant leading to a failure in the activation of the adaptive immune response. In this study, the transcriptomic profiles of monocyte-derived macrophages (MDM), isolated from four immunosuppressed kidney transplant patients and exposed to SARS-CoV-2 VOCs, were analyzed and compared with previously published data gathered from immune-competent subjects. Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of four kidney transplant patients admitted to the IRCCS Policlinico San Matteo of Pavia (Italy), differentiated into macrophages, and exposed to the active and the UV-inactivated particles of the different SARS-CoV-2 VOCs (D614G, Alpha B.1.1.7, Gamma P.1, Delta B.1.617.2 and Omicron BA.1). Bulk RNA-Seq was performed and significant transcripts were assessed based on Student’s t-test (p-value < 0.05) and Fold change > 2. RNA-Seq data analyses of immunosuppressed MDMs showed that SARS-CoV-2 VOCs, although transcriptionally active, did not induce strong alterations in the transcriptomic profiles of these cells, while a strong down-regulation of key genes involved in the innate immunity pathways was observed when comparing these data to the ones obtained from immunocompetent participants. Overall, this study suggests that patients under immunosuppressive therapies do have an altered macrophage response to SARS-CoV-2 viral infection.

End-stage liver disease (ESLD), affecting millions worldwide, represents a challenging issue for clinical research and global public health. Liver transplantation is the gold standard therapeutic approach but shows some drawbacks. Hepatocyte transplantation could be a reliable alternative for patient treatment. Mesenchymal stromal cells derived from Wharton’s jelly of the umbilical cord (WJ-MSCs) can differentiate into hepatocyte-like cells (HLCs) and show immunomodulatory functions. Due to the increasing demand for fully characterized cell therapy vehicles warranting both the safety and efficacy of treatments, in this work, we extensively characterized WJ-MSCs before and after the application of a hepatocyte-directed differentiation protocol. HLCs exhibited a morphology resembling that of hepatocytes, expressed early and late hepatic markers (α-fetoprotein, albumin, CK18, HNF4-α), and acquired hepatic functions (glycogen synthesis, xenobiotics detoxification), as also revealed by the shotgun proteomics approach. HLCs maintained the same pattern of immunomodulatory molecule expression and mesenchymal markers, other than displaying specific enzymes, suggesting these cells as promising candidates for cellular therapy of ESLD. Our work shed new light on the basic biology of HLCs, suggesting new therapeutic approaches to treat ESLD.