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Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune responseactivating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice. [ABSTRACT FROM AUTHOR]

Simple Summary: Cell–cell communication mechanisms are gathering growing scientific interest, particularly in the context of cancer cells and the tumor microenvironment. Extracellular vesicles are gaining increased interest due to their relevance in tumor molecular characterization, classification, diagnosis, prognosis evaluation, and response to treatment. Many advances have been made in the clinical and therapeutic fields, exploiting increasingly precise biomolecular engineering strategies. This review aims to focus on the role of extracellular vesicles (EVs) as diagnostic and therapeutic tools in lung cancer. Lung cancer represents the leading cause of cancer-related mortality worldwide, with around 1.8 million deaths in 2020. For this reason, there is an enormous interest in finding early diagnostic tools and novel therapeutic approaches, one of which is extracellular vesicles (EVs). EVs are nanoscale membranous particles that can carry proteins, lipids, and nucleic acids (DNA and RNA), mediating various biological processes, especially in cell–cell communication. As such, they represent an interesting biomarker for diagnostic analysis that can be performed easily by liquid biopsy. Moreover, their growing dataset shows promising results as drug delivery cargo. The aim of our work is to summarize the recent advances in and possible implications of EVs for early diagnosis and innovative therapies for lung cancer. [ABSTRACT FROM AUTHOR]

Pancreatic cancer (PCa) is the fifth leading cause of cancer mortality. Recently, our group and others have demonstrated the oncolytic activity of the Zika virus (ZIKV) against glioblastoma. The peculiar features of this virus offer the opportunity to use an agent already tested in vivo through natural transmission, with minimal effects on adults, to specifically target a tumor such as glioblastoma. This remarkable specificity prompted us to explore the potential use of ZIKV oncolytic action against other tumor types. In particular, we focused on the subgroup of pancreatic tumors with a neuroendocrine origin known as neuroendocrine tumors (NETs). We found that ZIKV exerts its oncolytic activity by specifically infecting NET cells, leading to growth inhibition and cell death. We also assessed whether the oncolytic action could be extended to pancreatic tumors different from NETs. However, as expected, the viral specificity is limited to NETs and is not applicable to adenocarcinoma tumors, indicating a narrow spectrum of action for this virus. These findings support the potential use of ZIKV in therapeutic approaches not only in glioblastoma, but also against other tumors, such as neuroendocrine pancreatic tumors. [ABSTRACT FROM AUTHOR]

The potential of perinatal tissues to provide cellular populations to be used in different applications of regenerative medicine is well established. Recently, the efforts of researchers are being addressed regarding the evaluation of cell products (secreted molecules or extracellular vesicles, EVs) to be used as an alternative to cellular infusion. The data regarding the effective recapitulation of most perinatal cells' properties by their secreted complement point in this direction. EVs secreted from perinatal cells exhibit key therapeutic effects such as tissue repair and regeneration, the suppression of inflammatory responses, immune system modulation, and a variety of other functions. Although the properties of EVs from perinatal derivatives and their significant potential for therapeutic success are amply recognized, several challenges still remain that need to be addressed. In the present review, we provide an up-to-date analysis of the most recent results in the field, which can be addressed in future research in order to overcome the challenges that are still present in the characterization and utilization of the secreted complement of perinatal cells and, in particular, mesenchymal stromal cells. [ABSTRACT FROM AUTHOR]

Neural stem cells (NSCs) were described for the first time more than two decades ago for their ability to differentiate into all neural cell lineages. The isolation of NSCs from adults and embryos was carried out by various laboratories and in different species, from mice to humans. Similarly, no more than two decades ago, cancer stem cells were described. Cancer stem cells, previously identified in hematological malignancies, have now been isolated from several solid tumors (breast, brain, and gastrointestinal compartment). Though the origin of these cells is still unknown, there is a wide consensus about their role in tumor onset, propagation and, in particular, resistance to treatments. Normal and neoplastic neural stem cells share common characteristics, and can thus be considered as two sides of the same coin. This is particularly true in the case of the Zika virus (ZIKV), which has been described as an inhibitor of neural development by specifically targeting NSCs. This understanding prompted us and other groups to evaluate ZIKV action in glioblastoma stem cells (GSCs). The results indicate an oncolytic activity of this virus vs. GSCs, opening potentially new possibilities in glioblastoma treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: With the aim of improving the therapeutic potential of mesenchymal stromal/stem cells (MSCs), we analyzed miRNA expression to investigate the effects of priming on intracellular and exosome (EXO)-derived miRNAs of MSCs. We primed MSCs with 3D culture (3D MSCs) or IFN-γ treatment (γ-MSCs), and EXOs were isolated from the conditioned medium. The miRNA analysis revealed similar expression patterns in intracellular miRNAs among biological replicates, while we observed noticeable variability in EXO miRNAs released even with the same priming condition. Although the MSCs and their EXOs exhibited distinct miRNA profiles following each priming treatment, we found deregulated miRNAs in common between the two sample types. The gene ontology of the deregulated miRNAs obtained after priming showed that MSC and EXO-derived miRNAs were functionally associated with tissue repair/regeneration. Specifically, γ-MSCs, 3D MSC EXOs, and γ-MSC EXOs contained more enriched miRNAs related to immunomodulation compared with 3D MSCs. Moreover, compared with IFN-γ treatment, both cells and EXOs derived from the 3D culture had more enriched miRNAs targeting genes involved in angiogenesis. Our study demonstrates that both 3D culture and IFN-γ treatment are able to modify intracellular and exosomal miRNAs, and our findings might contribute to a better understanding of the molecular mechanisms underlying the miRNA-mediated beneficial effects of MSCs. Mesenchymal stromal/stem cells (MSCs) have emerged as a therapeutic tool in regenerative medicine. Recent studies have shown that exosome (EXO)-derived microRNAs (miRNAs) play a crucial role in mediating MSC functions. Additionally, intracellular miRNAs have been found to regulate MSC therapeutic capacities. However, the molecular mechanisms underlying miRNA-mediated MSC effects are not fully understood. We used 3D culture and IFN-γ to prime/enhance the MSC therapeutic effects in terms of functional miRNAs. After priming, our analysis revealed stable variations in intracellular miRNA among the MSC biological replicates. Conversely, a significant variability of miRNA was observed among EXOs released from biological replicates of the priming treatment. For each priming, we observed distinct miRNA expression profiles between the MSCs and their EXOs. Moreover, in both types of priming, gene ontology (GO) analysis of deregulated miRNAs highlighted their involvement in tissue repair/regeneration pathways. In particular, the 3D culture enhanced angiogenic properties in both MSCs and EXOs, while IFN-γ treatment enriched miRNAs associated with immunomodulatory pathways. These findings suggest that 3D culture and IFN-γ treatment are promising strategies for enhancing the therapeutic potential of MSCs by modulating miRNA expression. Additionally, the identified miRNAs may contribute to understanding the molecular mechanisms underlying the miRNA-mediated therapeutic effects of MSCs. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which counts more than 650 million cases and more than 6.6 million of deaths worldwide, affects the respiratory system with typical symptoms such as fever, cough, sore throat, acute respiratory distress syndrome (ARDS), and fatigue. Other nonpulmonary manifestations are related with abnormal inflammatory response, the "cytokine storm", that could lead to a multiorgan disease and to death. Evolution of effective vaccines against SARS-CoV-2 provided multiple options to prevent the infection, but the treatment of the severe forms remains difficult to manage. The cytokine storm is usually counteracted with standard medical care and anti-inflammatory drugs, but researchers moved forward their studies on new strategies based on cell therapy approaches. The perinatal tissues, such as placental membranes, amniotic fluid, and umbilical cord derivatives, are enriched in mesenchymal stromal cells (MSCs) that exert a well-known anti-inflammatory role, immune response modulation, and tissue repair. In this review, we focused on umbilical-cord-derived MSCs (UC-MSCs) used in in vitro and in vivo studies in order to evaluate the weakening of the severe symptoms, and on recent clinical trials from different databases, supporting the favorable potential of UC-MSCs as therapeutic strategy. [ABSTRACT FROM AUTHOR]

Ischemia/reperfusion injury (IRI) is a multistep damage that occurs in several tissues when a blood flow interruption is inevitable, such as during organ surgery or transplantation. It is responsible for cell death and tissue dysfunction, thus leading, in the case of transplantation, to organ rejection. IRI takes place during reperfusion, i.e., when blood flow is restored, by activating inflammation and reactive oxygen species (ROS) production, causing mitochondrial damage and apoptosis of parenchymal cells. Unfortunately, none of the therapies currently in use are definitive, prompting the need for new therapeutic approaches. Scientific evidence has proven that mesenchymal stem/stromal cells (MSCs) can reduce inflammation and ROS, prompting this cellular therapy to also be investigated for treatment of IRI. Moreover, it has been shown that MSC therapeutic effects were mediated in part by their secretome, which appears to be involved in immune regulation and tissue repair. For these reasons, mediated MSC paracrine function might be key for injury amelioration upon IRI damage. In this review, we highlight the scientific literature on the potential beneficial use of MSCs and their products for improving IRI outcomes in different tissues/organs, focusing in particular on the paracrine effects mediated by MSCs, and on the molecular mechanisms behind these effects. [ABSTRACT FROM AUTHOR]

At present, there is a lack of clinical evidence about the impact and long-term durability of the immune response induced by the third dose of mRNA vaccines. In this study, we followed up the B cell compartment behavior in a cohort of immunocompetent individuals three and six months after the third dose of vaccine. During this period, some subjects contracted the virus. In uninfected vaccinated subjects, we did not report any changes in serum spike-specific IgG levels, with a significant reduction in IgA. Instead, subjects recovered from natural infection showed a significant increase in both specific IgG and IgA. Moreover, we showed a time-related decrease in IgG neutralizing potential to all SARS-CoV-2 variants of concern (VOC) in uninfected compared to recovered subjects, who displayed an increased neutralizing ability, particularly against the omicron variant. Finally, we underlined the presence of a pool of SARS-CoV-2-specific B cells in both groups that are prone to respond to restimulation, as demonstrated by their ability to differentiate into plasma cells and to produce anti-SARS-CoV-2-specific immunoglobulins. These data lead us to assert the long-term effectiveness of the BNT162b2 vaccine in contrasting the severe form of the pathology and prevent COVID-19-associated hospitalization. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) constitute one of the main mechanisms by which cells communicate with the surrounding tissue or at distance. Vesicle secretion is featured by most cell types, and adult mesenchymal stromal cells (MSCs) of different tissue origins have shown the ability to produce them. In recent years, several reports disclosed the molecular composition and suggested clinical indications for EVs derived from adult MSCs. The parental cells were already known for their roles in different disease settings in regulating inflammation, immune modulation, or transdifferentiation to promote cell repopulation. Interestingly, most reports also suggested that part of the properties of parental cells were maintained by isolated EV populations. This review analyzes the recent development in the field of cell-free therapies, focusing on several adult tissues as a source of MSC-derived EVs and the available clinical data from in vivo models. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is modifying human activity all over the world with significant health and economic burden. The advent of the SARS-CoV-2 pandemic prompted the scientific community to learn the virus dynamics concerning transmissibility, epidemiology, and usefulness of vaccines in fighting emerging health hazards. Pieces of evidence suggest that the first and second doses of mRNA vaccines induce a significant antibody response in vaccinated subjects or patients who recovered from SARS-CoV-2 infection, demonstrating the importance of the previously formed memory. The aim of this work has been to investigate the effects of BNT162b2 Pfizer-BioNTech mRNA-based vaccine booster dose in a cohort of 11 uninfected immunocompetent (ICs), evaluating the humoral and cellular responses, with more carefulness on memory B and T cells. Our findings underscore the potential benefit of the third dose of mRNA vaccine on the lifespan of memory B and T cells, suggesting that booster doses could increase protection against SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Primary graft dysfunction (PGD) and ischemia-reperfusion injury (IRI) occur in up to 30% of patients undergoing lung transplantation and may impact on the clinical outcome. Several strategies for the prevention and treatment of PGD have been proposed, but with limited use in clinical practice. In this study, we investigate the potential application of sevoflurane (SEV) preconditioning to mitigate IRI after lung transplantation. The study included two groups of swines (preconditioned and not preconditioned with SEV) undergoing left lung transplantation after 24-hour of cold ischemia. Recipients' data was collected for 6 hours after reperfusion. Outcome analysis included assessment of ventilatory, hemodynamic, and hemogasanalytic parameters, evaluation of cellularity and cytokines in BAL samples, and histological analysis of tissue samples. Hemogasanalytic, hemodynamic, and respiratory parameters were significantly favorable, and the histological score showed less inflammatory and fibrotic injury in animals receiving SEV treatment. BAL cellular and cytokine profiling showed an anti-inflammatory pattern in animals receiving SEV compared to controls. In a swine model of lung transplantation after prolonged cold ischemia, SEV showed to mitigate the adverse effects of ischemia/reperfusion and to improve animal survival. Given the low cost and easy applicability, the administration of SEV in lung donors may be more extensively explored in clinical practice. [ABSTRACT FROM AUTHOR]

The awareness of viruses as a constant threat for human public health is a matter of fact and in this resides the need of understanding the mechanisms they use to trick the host. Viral non-coding RNAs are gaining much value and interest for the potential impact played in host gene regulation, acting as fine tuners of host cellular defense mechanisms. The implicit importance of v-ncRNAs resides first in the limited genomes size of viruses carrying only strictly necessary genomic sequences. The other crucial and appealing characteristic of v-ncRNAs is the non-immunogenicity, making them the perfect expedient to be used in the never-ending virus-host war. In this review, we wish to examine how DNA and RNA viruses have evolved a common strategy and which the crucial host pathways are targeted through v-ncRNAs in order to grant and facilitate their life cycle. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles have been used effectively in different lung disease animal models and clinical trials. Their specific beneficial effects, the potential differences between MSCs derived from different organs, and interactions between MSC products and target cells still need to be studied further. Therefore, we investigated the effects of secreted products of human MSCs derived from the bone marrow and adipose tissue on human lung small airway epithelial (AE) cells in vitro. AE cells were cocultured with MSCs in inserts that allowed the free exchange of medium but did not allow direct cell-to-cell contact. We examined the effects on AE cell viability, proliferation, cell numbers, expression of AE cell-specific genes, and CD54 (intercellular adhesion molecule 1 (ICAM1)) surface positivity, as well as the secretion/uptake of growth factors relevant for AE cell. We found that coculture increased the viability of AE cells. The majority of AE cells expressed CD54 on their surface, but the percentage of cells being positive for CD54 did not increase in coculture. However, ICAM1 gene expression was increased in coculture. Also, we observed increased gene expression of mucin (MUC1), a lung-enriched cell surface glycoprotein. These observed effects were the same between bone marrow and adipose tissue MSCs. However, MSCs derived from adipose tissue reduced angiopoietin concentrations in coculture, whereas those from the bone marrow did not. Conclusively, MSCs influenced AE cells positively by increasing their viability and affecting gene expression, with some effects being specific for the tissue origin of MSCs. [ABSTRACT FROM AUTHOR]

Human mesenchymal stromal/stem cells (MSCs), being immunoprivileged and having immunomodulatory ability, represent a promising tool to be applied in the field of regenerative medicine. Based on numerous in vitro evidences, the immunological effects of MSCs on immune cells could depend on different mechanisms as cell-to-cell contact and paracrine signals. Furthermore, recent studies have shown that the immunomodulatory activity of MSCs is initiated by activated immune cells; thus, their interaction represents a potential homeostatic mechanism by which MSCs regulate the immune response. MSCs also release exosomes able to give different effects, in a paracrine manner, by influencing inflammatory processes. In this study, we aimed to establish the potential role of human amnion-derived MSCs (hAMSCs), in immunomodulation. We found that the immunosuppressive properties of hAMSCs are not constitutive, but require "supportive signals" capable of promoting these properties. Indeed, we observed that hAMSCs alone are not able to produce an adequate amount of soluble immunomodulatory factors. Here, we studied, in depth, the strong immunomodulatory licensing signal deriving from the direct interaction between hAMSCs and stimulated peripheral blood mononuclear cells. We found that the immunomodulatory effect of hAMSCs also depends on cell-to-cell contact through the contribution of the PDL-1/PD-1 axis. We then investigated the IFN-γ priming of hAMSCs (γ-hAMSCs), which induce the increase of PDL-1 expression, high production of IDO, and upregulation of different immunomodulatory exosome-derived miRNAs. Our miRNA–target network analysis revealed that nine of the deregulated miRNAs are involved in the regulation of key proteins that control both T cell activation/anergy and monocyte differentiation pathways. Finally, we observed that γ-hAMSCs induce in monocytes both M2-like phenotype and the increase of IL-10 production. The extensive implications of MSCs in modulating different aspects of the immune system make these cells attractive candidates to be employed in therapeutic application in immune-based diseases. For these reasons, we aimed, with this study, to shed light on the potential of hAMSCs, and how they could become a useful tool for treating different inflammatory diseases, including end-stage pathologies or adverse effects in transplanted patients. [ABSTRACT FROM AUTHOR]

Different approaches have been studied in both preclinical and clinical settings to develop cell-based therapies and/or engineered cell-based therapies to better integrate grafts with the host. In these techniques, much attention is addressed to the use of adult stem cells such as mesenchymal stem cells (MSCs), but identifying and obtaining sufficient numbers of therapeutic cells, and the right route of administration, is often a challenge. In this study, we tested the feasibility of encapsulating human amnion-derived MSCs (hAMSCs) in a semipermeable and biocompatible fiber as a new approach for regenerative medicine. Our data showed that hAMSCs aggregated in the device constitutes an effective system for enhancing, or at least for maintaining, the paracrine activity of these cells in order to better promote tissue regeneration in an immune isolated state. In our new experimental approach, the hAMSCs retained their therapeutic potential, as shown by both the production of specific immunomodulatory/angiogenic factors and immunomodulatory and angiogenic ability observed in vitro. Unlike cell infusion methods, the use of encapsulated-cells leads to minimally invasive approaches, avoiding a direct interaction with the host. Therefore, the potentiality of an allograft or xenograft without the need for immunosuppression, and the lack of tumorigenesis is very intriguing. • Human amnion-derived MSCs (hAMSCs) secrete a panel of factors related to tissue repair. • hAMSCs encapsulated in a semipermeable device retained their secretory activity. • Device-encapsulated hAMSCs may represent a new approach for regenerative medicine avoiding allogenic-related problems. [ABSTRACT FROM AUTHOR]

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies. [ABSTRACT FROM AUTHOR]

Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure. [ABSTRACT FROM AUTHOR]

The product of neurofibromatosis type 2 (NF2) gene, also known as Merlin/neurofibromin 2, homeostatically regulates liver stem cells by controlling abundance and signaling of epidermal growth factor receptor (EGFR), with a mechanism independent of the Hippo pathway. We have reported that locally elevated estrogen formation, driven by abnormally high expression and function of aromatase, may be implicated in development and progression of human hepatocellular carcinoma (HCC) through activation of a rapid signaling pathway mediated by amphiregulin (AREG) and EGFR. We have recently presented a model by which the aromatase-estrogen-amphiregulin-EGFR axis is activated in response to tissue injury and/or inflammatory disease, with its alteration eventually leading to development of major human tumors (liver, breast, prostate) and other chronic diseases (diabetes, obesity, Alzheimer’s and heart disease). In this study, we investigated NF2 expression in liver cancer cells and tissues in relation to aromatase expression/function, estrogen receptor (ER) status and amphiregulin. Our data indicate that NF2 expression is associated with aromatase and AREG expression, being elevated in HCC tissues and HepG2 cells, intermediate in cirrhotic tissues and Huh7 cells, and lower in nontumoral liver and HA22T cells. In addition, NF2 expression is inversely related to wild type hERα66 and proportional to the expression of the membrane-associated hERα36 splice variant, as measured by exon-specific RT-PCR analysis, both in vivo and in vitro . Furthermore, incubation with estradiol induced a significant decrease of NF2 expression in both HA22T and Huh7 cells (over 54% and 22%, respectively), while no change could be observed in HepG2 cells, this effect being inversely related to aromatase expression and activity in HCC cell lines. Based on the above combined evidence, we hypothesize that NF2 behaves as a protein sensing tissue damage and aromatase-driven local estrogen formation, eventually leading to regulation of stem cells differentiation and tissue repair. [ABSTRACT FROM AUTHOR]

Lung transplantation (LTx) has become the gold standard treatment for end-stage respiratory failure. Recently, extended lung donor criteria have been applied to decrease the mortality rate of patients on the waiting list. Moreover, ex vivo lung perfusion (EVLP) has been used to improve the number/quality of previously unacceptable lungs. Despite the above-mentioned progress, the morbidity/mortality of LTx remains high compared to other solid organ transplants. Lungs are particularly susceptible to ischemia-reperfusion injury, which can lead to graft dysfunction. Therefore, the success of LTx is related to the quality/function of the graft, and EVLP represents an opportunity to protect/regenerate the lungs before transplantation. Increasing evidence supports the use of mesenchymal stromal/stem cells (MSCs) as a therapeutic strategy to improve EVLP. The therapeutic properties of MSC are partially mediated by secreted factors. Hence, the strategy of lung perfusion with MSCs and/or their products pave the way for a new innovative approach that further increases the potential for the use of EVLP. This article provides an overview of experimental, preclinical and clinical studies supporting the application of MSCs to improve EVLP, the ultimate goal being efficient organ reconditioning in order to expand the donor lung pool and to improve transplant outcomes. [ABSTRACT FROM AUTHOR]

Ischemia/reperfusion injury (IRI) represents one of the leading causes of primary non-function acute liver transplantation failure. IRI, generated by an interruption of organ blood flow and the subsequent restoration upon transplant, i.e., reperfusion, generates the activation of an inflammatory cascade from the resident Kupffer cells, leading first to neutrophils recruitment and second to apoptosis of the parenchyma. Recently, human mesenchymal stromal/stem cells (hMSCs) and derivatives have been implemented for reducing the damage induced by IRI. Interestingly, sparse data in the literature have described the use of human amnion-derived MSCs (hAMSCs) and, more importantly, no evidence regarding hMSCs priming on liver IRI have been described yet. Thus, our study focused on the definition of an in vitro model of liver IRI to test the effect of primed hAMSCs to reduce IRI damage on immune and hepatic cells. We found that the IFNγ pre-treatment and 3D culture of hAMSCs strongly reduced inflammation induced by M1-differentiated macrophages. Furthermore, primed hAMSCs significantly inhibited parenchymal apoptosis at early timepoints of reperfusion by blocking the activation of caspase 3/7. All together, these data demonstrate that hAMSCs priming significantly overcomes IRI effects in vitro by engaging the possibility of defining the molecular pathways involved in this process. [ABSTRACT FROM AUTHOR]

Several studies suggest that xanthine dehydrogenase (XDH) and its oxidase form (XO) play an important role in various types of ischemic and vascular injuries. Recently, we have demonstrated that estradiol (E2) induces a significant decrease of the expression and activity of XDH and of its conversion to XO in human mammary epithelial cells. E2 is known to induce upregulation of eNOS gene expression in aortic endothelial cells. Because the XO-derived O2·- combines with ·NO to yield ONOO-, and considering that ONOO- converts XDH to XO, the resulting increase of XO activity and reactive oxygen species production would eventually lead to a further increase of ONOO- production, thus creating a vicious cycle of oxidative stress. Our previous study has indicated that sildenafil has a protective effect on human mammary epithelial cells as a consequence of XO inhibition and of the resulting decrease of free oxygen radicals that can impair the expression of NADPH oxidase and type 5 phosphodiesterase (PDE-5). In the present study, we report that the dual inhibitory effect exerted by sildenafil on both XO and PDE-5 is a consequence of a structural modification induced by O2·-, also consisting of the release of a piperazine group that could in turn inhibit the XO enzyme. [ABSTRACT FROM AUTHOR]

Although estrogen receptors (ERs) are expressed in human hepatocellular carcinoma (HCC), several clinical trials have failed to demonstrate the efficacy of antiestrogen treatment in HCC patients. Recently, the identification of several ER splicing variants has enlightened the complex nature of estrogen signaling in peripheral tissues; this may help understanding estrogen role in either nontumoral or malignant nonclassical target organs, including liver. In this work we have investigated mRNA expression of wild-type and splice variants of ERα in nontumoral, cirrhotic, and malignant human liver, as well as in HCC cell lines, using an exon-specific reverse transcription polymerase chain reaction (RT-PCR). In particular, ERα66 was detected in nontumoral and, to a lesser extent, in cirrhotic liver tissues, whereas its expression decreased or became undetectable in HCC tissues and cell lines. The ERα46 splicing variant was detected ubiquitously in all samples; interestingly, however, the ERα36 variant was inversely expressed with respect to ERα66, being highest in HepG2 cells, intermediate in Huh7 cells, and lowest in HA22T cells. It is noteworthy that aromatase was correspondingly expressed with ERα36 and inversely related to ERα66. This observation suggests that a switch from ERα66 to a predominant expression of ERα36 may be associated with development and/or progression of human HCC. [ABSTRACT FROM AUTHOR]

Background: We investigated aromatase (Aro)-driven estrogen formation in non-tumoral and malignant liver tissues and cells, also in relation to expression of the estrogen receptors α and β (ERα and ERβ) and amphiregulin (AREG), aiming to gain insights into the potential role of estrogens in human hepatocellular carcinoma (HCC). Materials and methods: Chromatographic and reverse transcriptase polymerase chain reaction (RT-PCR) analyses were used to assess activity and expression of the Aro enzyme and AREG as well as the expression of wild-type and variant ERs, both in vivo and in vitro. Results: Following 24 h and 72 h incubation of liver tissues or cells with testosterone, human HCC tissues and HepG2 hepatoma cells showed elevated Aro activity (estrogen formation, respectively, of 20% and 52%-99%). By contrast, no Aro activity could be detected in non-tumoral tissues and HA22T liver cancer cells. Cirrhotic samples and Huh7 cells exhibited intermediate enzyme activity, with estrogen formation of 4% and 34%, respectively. Markedly lower or undetectable Aro mRNA levels were observed in HA22T cells and non-tumoral liver tissues compared with HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels. Interestingly, no or low expression of wild-type ERα and ERβ could be observed in liver cancer cells and malignant tissues. However, ubiquitous expression of the hERα46 variant and occasional expression of the hERβ2/Cx variant were observed in cancer tissues and cells. Conclusions: It is noteworthy that the pattern of wild-type ERα was inversely related to Aro, whilst AREG expression was consistently associated with that of Aro. This combined evidence suggests that locally elevated Aro activity may increase malignant cell proliferation also through AREG signalling. [ABSTRACT FROM AUTHOR]

Xanthine oxidase (XO) plays an important role in various forms of ischemic and vascular injuries, inflammatory diseases and chronic heart failure. The XO inhibitors allopurinol and oxypurinol held considerable promise in the treatment of these conditions both in experimental animals and in human clinical trials. More recently, an endothelium-based protective effect of sildenafil has been reported in preconditioning prior to ischemia/reperfusion in healthy human subjects. Based on the structural similarities between allopurinol and oxypurinol with sildenafil and with zaprinast the authors have investigated the potential effects of these latter compounds on the buttermilk XO and on non-tumourigenic (HMEC) and malignant (MCF7) human mammary epithelial cells. Both sildenafil and zaprinast induced a significant and consistent decrease of XO expression and activity in either cell line. In MCF7 cells only, this effect was associated with the abrogation of xanthineinduced cytotoxicity. Overall, the data suggest that the protective effect of sildenafil on epithelial cells is a consequence of the inhibition of the XO and of the resulting decrease of free oxygen radical production that may influence the expression of NADPH oxidase and PDE-5. [ABSTRACT FROM AUTHOR]

Human hepatocellular carcinoma (HCC) is associated with high mortality rates, being the third most common cause of cancer death worldwide. Although estrogens have been implicated in HCC, their potential role in development and/or progression of this malignancy remains unclear. In this study we investigated mRNA and protein expression of aromatase (Aro) and amphiregulin (AREG) in relation to estrogen receptors (ERs), in HepG2, Huh7, and HA22T human malignant liver cell lines, using RT-PCR and Western blot analyses. Aro expression was significantly higher (∼13-fold, P= 0.003) in HepG2 cells than in Huh7 cells, while no Aro expression could be detected in HA22T cells. Interestingly, the patterns of AREG expression were consistently associated with those of Aro, with ∼3-fold and ∼8-fold higher levels being seen in HepG2 cells than in Huh7 cells ( P= 0.002) and HA22T cells ( P= 0.0014), respectively. Using a specific set of primers, ERα46 is the only ER variant expressed in all cell lines, while the wild-type ERα66 could not be detected. Western blot analysis revealed a corresponding figure. This evidence suggests that AREG expression may be upregulated by estrogens in human HCC and that locally elevated aromatase activity also may increase malignant cell proliferation through AREG signaling. [ABSTRACT FROM AUTHOR]

In this study, we investigated androgen metabolism in two different human prostate cancer cell lines, the androgen-responsive LNCaP cells and the nonresponsive PC3 cells. Following 24-h and 72-h incubation with either testosterone (T) or androstenedione (Ad) used as precursor, divergent patterns and rates of androgen metabolism were observed. Given the recent interest in the multiple uses of embryonic and adult stem cells for basic and applied research, we compared the expression of three presumptive stem cell markers (Oct-4, SUZ-12, and Cripto-1), along with connexin 43 (Cx43), Cx32, and androgen receptor (AR), used as cell differentiation gene markers. In anchorage-independent cell growth conditions, the expression levels of candidate markers of cancer stem cells initially increased (days 2–4) but drastically fell thereafter (day 6) in both cell lines. Results of immunocytochemical assay (ICA) largely confirmed those obtained by RT-PCR. Interestingly, both symmetrical and asymmetrical cell divisions were revealed in PC3 cells using Oct-4 immunostaining. Our data suggest that both androgen-responsive and androgen-nonresponsive prostate tumor cell lines contain a presumptive cancer stem cell population that can be identified using a panel of selected gene markers, including Oct-4, SUZ-12, and Cripto-1. [ABSTRACT FROM AUTHOR]

A new classification based on gene expression profiling or immunohistochemical (IHC) characteristics may replace current histopathological classifications and predict better clinical outcomes. We used IHC markers to classify incident cases ascertained by the Palermo Breast Cancer Registry (2002–2004) into four subtypes: luminal-A (ER+ or PgR+ and HER2/neu−); luminal-B (ER+ or PgR+, HER2/neu+); basal-like (ER−, PgR−, HER2/neu−); and HER2+/ER− (HER2/neu+, ER−, PgR−). We evaluated HER2/neu, ER and PgR in 1300/1985 (65%) cases. The most common IHC-subtype was luminal-A (68%), whereas luminal-B, basal-like, and HER2+/ER− accounted for 14%, 13%, and 5%, respectively. IHC-subtypes were not associated with tumor size, geographic location within the province, or menopause, but differed by NPI ( P < 0.0001), grading ( P < 0.0001), lymph-node involvement ( P= 0.04), metastases ( P= 0.04), and TNM stage ( P= 0.04). Endocrine therapy was administered to 81% of 519 postmenopausal, luminal-A, and luminal-B cases and to 32% of 114 postmenopausal, basal-like, and HER2+/ER− cases. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine. [ABSTRACT FROM AUTHOR]

The use of extracorporeal membrane oxygenation (ECMO) for acute respiratory distress syndrome (ARDS) has increased in the last decade. However, mortality remains high, and the complexity of ECMO requires individualized treatment. There are some biomarkers to monitor progression and predict clinical outcomes of ARDS. This project aims to advance the management of ARDS patients treated with ECMO by exploring miRNA expression in whole blood. The analysis was conducted on two groups with different length of ECMO: Group A (longer runs) and group B (shorter runs). We analyzed miRNAs before ECMO cannulation, and at 7 and 14 days of ECMO support. Our results showed that in the group B patients, 11 deregulated miRNAs were identified, and showed an opposite trend of expression compared to the group A patients. In silico analysis revealed that these 11 miRNAs were related to processes involved in the pathogenesis and evolution of ARDS. This scenario could represent homeostatic mechanisms by which, in ECMO responsive patients, pathways activated during ARDS progression are switched-off. Circulating miRNAs could represent promising biomarkers to monitor the evolution of ARDS under ECMO support. Further studies may shed light on this topic to improve a personalized approach in such a complex setting of patients. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal/stem cells (MSCs) are multipotent adult stem cells that support homeostasis during tissue regeneration. In the last decade, cell therapies based on the use of MSCs have emerged as a promising strategy in the field of regenerative medicine. Although these cells possess robust therapeutic properties that can be applied in the treatment of different diseases, variables in preclinical and clinical trials lead to inconsistent outcomes. MSC therapeutic effects result from the secretion of bioactive molecules affected by either local microenvironment or MSC culture conditions. Hence, MSC paracrine action is currently being explored in several clinical settings either using a conditioned medium (CM) or MSC-derived exosomes (EXOs), where these products modulate tissue responses in different types of injuries. In this scenario, MSC paracrine mechanisms provide a promising framework for enhancing MSC therapeutic benefits, where the composition of secretome can be modulated by priming of the MSCs. In this review, we examine the literature on the priming of MSCs as a tool to enhance their therapeutic properties applicable to the main processes involved in tissue regeneration, including the reduction of fibrosis, the immunomodulation, the stimulation of angiogenesis, and the stimulation of resident progenitor cells, thereby providing new insights for the therapeutic use of MSCs-derived products. [ABSTRACT FROM AUTHOR]

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

There is a need to improve acute respiratory distress syndrome (ARDS) diagnosis and management, particularly with extracorporeal membrane oxygenation (ECMO), and different biomarkers have been tested to implement a precision-focused approach. We included ARDS patients on veno-venous (V-V) ECMO in a prospective observational pilot study. Blood samples were obtained before cannulation, and screened for the expression of 754 circulating microRNA (miRNAs) using high-throughput qPCR and hierarchical cluster analysis. The miRNet database was used to predict target genes of deregulated miRNAs, and the DIANA tool was used to identify significant enrichment pathways. A hierarchical cluster of 229 miRNAs (identified after quality control screening) produced a clear separation of 11 patients into two groups: considering the baseline SAPS II, SOFA, and RESP score cluster A (n = 6) showed higher severity compared to cluster B (n = 5); p values < 0.05. After analysis of differentially expressed miRNAs between the two clusters, 95 deregulated miRNAs were identified, and reduced to 13 by in silico analysis. These miRNAs target genes implicated in tissue remodeling, immune system, and blood coagulation pathways. The blood levels of 13 miRNAs are altered in severe ARDS. Further investigations will have to match miRNA results with inflammatory biomarkers and clinical data. [ABSTRACT FROM AUTHOR]

The theory that viruses play a role in human cancers is now supported by scientific evidence. In fact, around 12% of human cancers, a leading cause of morbidity and mortality in some regions, are attributed to viral infections. However, the molecular mechanism remains complex to decipher. In recent decades, the uncovering of cellular miRNAs, with their invaluable potential as diagnostic and prognostic biomarkers, has increased the number of studies being conducted regarding human cancer diagnosis. Viruses develop clever mechanisms to succeed in the maintenance of the viral life cycle, and some viruses, especially herpesviruses, encode for miRNA, v-miRNAs. Through this viral miRNA, the viruses are able to manipulate cellular and viral gene expression, driving carcinogenesis and escaping the host innate or adaptive immune system. In this review, we have discussed the main viral miRNAs and virally influenced cellular pathways, and their capability to drive carcinogenesis. [ABSTRACT FROM AUTHOR]

Culturing fetal hepatocytes in high cell-density allowed stabilization of the hepatocyte phenotype up to 8 weeks, including the maintenance of liver-specific functions. On the other hand, when cultured at low cell-density, fetal hepatocytes underwent morphological modifications and acquired fibroblastic morphology. Since a switch from E-cadherin to vimentin expression accompanied these changes, we hypothesized the occurrence of epithelial-to-mesenchymal transition when fetal hepatocytes were cultured at low cell-density. Changes in gene expressionsuch as up-regulation of fibrosis-related geneswere also observed, suggesting that the low cell-density culture system promoted the acquisition of a profibrotic phenotype in cultured hepatocytes. The origin of fibrogenic cells in the liver is not well known, and the role of hepatocytes as a source of fibrogenic cells is controversial. Therefore, we hypothesized that hepatocytes undergoing epithelial-to-mesenchymal transition could have a central role in liver fibrosis as a source of fibrogenic cells. To conclude, the high cell-density culture system could be a useful model for in vitro studies requiring long-term cultures of hepatocytes, such as the development of pharmaceutical drugs and mechanisms of viral infections. The low cell-density culture system may provide additional insights into the origin of fibrogenic cells in the liver, thus contributing to the development of novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Abstract: There is indirect multiple evidence that hints at a potential role of sex steroids in development and progression of human hepatocellular carcinoma (HCC). In the present study, we have investigated androgen metabolism in a panel of human liver cancer cell lines (HA22T, Huh7, HepG2) and in normal, cirrhotic and malignant human liver tissues aiming to dissect the potential impact of individual enzyme activities and their products in normal and diseased human liver, both in vivo and in vitro. Using our intact cell analysis we were able to assess rates and pathways of androgen metabolism in living conditions. Overall, incubation of cultured cells or tissue minces with either testosterone (T) or androstenedione (Ad) used as precursor resulted in a large extent of 17βoxidation of T to Ad (cells: 28–77%; tissues: 35–50%). In malignant liver cell lines, both HA22T and Huh7 cells showed consistent amounts of the 5α-reductase enzyme products (18% and 15%, respectively), while 5β-reductase activity was more pronounced in Huh7 cells (18%) than in HA22T cells (1.8%). Interestingly, a significant extent of estrogen formation could be observed in Huh7 cells (5.4–11.5%), while no aromatase activity could be detected in HA22T cells. In HepG2 cells, along with a relatively high proportion of Ad, estrogens represented the most prominent (50–55%) end product of androgen metabolism, regardless of the precursor used. In liver tissues, equivalent results could be obtained, with a consistent proportion of 17βoxidation of T to Ad (35–50%) being observed in the majority of samples. However, while normal liver tissue samples exhibited a minor proportion of bioactive androgens (3.4%) with no aromatase products, HCC tissues showed a significant extent of aromatase activity (nearly 20%) with estrogen representing the most prominent metabolic product after 24h incubation with either T or Ad. HCV and alcoholic cirrhotic tissues displayed different patterns of androgen metabolism. The former produced limited amounts of bioactive androgens (5.3%) and considerable levels of the intermediate aromatase product 19OH-Ad (up to 28%), the latter exhibited a prevalence of androgen degradation through the 5β-reductase pathway (9.8%) and a significant extent of aromatase activity (16% as a whole). In conclusion, three major metabolic states could be depicted, depending on prevalent pathways of androgen metabolism and steroid receptor status: estrogenic, androgenic, and mixed. This model supports the idea that local estrogen biosynthesis may be implicated in human HCC and provides a basis for the exploitation of aromatase inhibitors and/or ER antagonists or selective estrogen receptor modulators (SERMs) as a new therapeutic strategy in HCC patients. [Copyright &y& Elsevier]

Mesenchymal stromal/stem cells (MSCs) have shown significant therapeutic potential, and have therefore been extensively investigated in preclinical studies of regenerative medicine. However, while MSCs have been shown to be safe as a cellular treatment, they have usually been therapeutically ineffective in human diseases. In fact, in many clinical trials it has been shown that MSCs have moderate or poor efficacy. This inefficacy appears to be ascribable primarily to the heterogeneity of MSCs. Recently, specific priming strategies have been used to improve the therapeutic properties of MSCs. In this review, we explore the literature on the principal priming approaches used to enhance the preclinical inefficacy of MSCs. We found that different priming strategies have been used to direct the therapeutic effects of MSCs toward specific pathological processes. Particularly, while hypoxic priming can be used primarily for the treatment of acute diseases, inflammatory cytokines can be used mainly to prime MSCs in order to treat chronic immune-related disorders. The shift in approach from regeneration to inflammation implies, in MSCs, a shift in the production of functional factors that stimulate regenerative or anti-inflammatory pathways. The opportunity to fine-tune the therapeutic properties of MSCs through different priming strategies could conceivably pave the way for optimizing their therapeutic potential., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Chronic hepatitis C virus (HCV) infection is the principal etiology of cirrhosis and, ultimately, hepatocellular carcinoma (HCC). At present, approximately 71 million people are chronically infected with HCV, and 10%-20% of these are expected to develop severe liver complications throughout their lifetime. Scientific evidence has clearly shown the causal association between miRNAs, HCV infection and HCC. Although it is not completely clear whether miRNA dysregulation in HCC is the cause or the consequence of its development, variations in miRNA patterns have been described in different liver diseases, including HCC. Many studies have analyzed the importance of circulating miRNAs and their effect on cell proliferation and apoptosis. In this Review, we aim to summarize current knowledge on the association between miRNA, HCV and HCC from a diagnostic point of view, and also the potential implications for therapeutic approaches., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflict of interest., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

The prevalence of end-stage liver diseases has reached very high levels globally. The election treatment for affected patients is orthotopic liver transplantation, which is a very complex procedure, and due to the limited number of suitable organ donors, considerable research is being done on alternative therapeutic options. For instance, the use of cell therapy, such as the transplantation of hepatocytes to promote liver repair/regeneration, has been explored, but standardized protocols to produce suitable human hepatocytes are still limited. On the other hand, liver progenitor and multipotent stem cells offer potential cell sources that could be used clinically. Different studies have reported regarding the therapeutic effects of transplanted mesenchymal stromal/stem cells (MSCs) on end-stage liver diseases. Moreover, it has been shown that delivery of MSC-derived conditioned medium (MSC-CM) can reduce cell death and enhance liver proliferation in fulminant hepatic failure. Therefore, it is believed that MSC-CM contains many factors that probably support liver regeneration. In our work, we used an in vitro model of human liver organoids to study if the paracrine components secreted by human amnion-derived MSCs (hAMSCs) affected liver stem/progenitor cell differentiation. In particular, we differentiated liver organoids derived from bipotent EpCAM + human liver cells and tested the effects of hAMSC secretome, derived from both two-dimensional (2D) and three-dimensional (3D) hAMSC cultures, on that model. Our analysis showed that conditioned medium (CM) produced by 3D hAMSCs was able to induce an over-expression of mature hepatocyte markers, such as ALB, NTCP, and CYP3A4, compared with both 2D hAMSC cultures and the conventional differentiation medium (DM). These data were confirmed by the over-production of ALB protein and over-activity of CYP3A4 observed in organoids grown in 3D hAMSC-CM. Liver repair dysfunction plays a role in the development of liver diseases, and effective repair likely requires the normal functioning of liver stem/progenitor cells. Herein, we showed that hAMSC-CM produced mainly by 3D cultures had the potential to increase hepatic stem/progenitor cell differentiation, demonstrating that soluble factors secreted by those cells are potentially responsible for the reaction. This work shows a potential approach to improve liver repair/regeneration also in a transplantation setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lo Nigro, Gallo, Bulati, Vitale, Paini, Pampalone, Galvagno, Conaldi and Miceli.)