Extracellular Vesicles as Delivery Vehicles for Non-Coding RNAs: Potential Biomarkers for Chronic Liver Diseases.

In recent years, EVs have emerged as promising vehicles for coding and non-coding RNAs (ncRNAs), which have demonstrated remarkable potential as biomarkers for various diseases, including chronic liver diseases (CLDs). EVs are small, membrane-bound particles released by cells, carrying an arsenal of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and other ncRNA species, such as piRNAs, circRNAs, and tsRNAs. These ncRNAs act as key regulators of gene expression, splicing, and translation, providing a comprehensive molecular snapshot of the cells of origin. The non-invasive nature of EV sampling, typically via blood or serum collection, makes them highly attractive candidates for clinical biomarker applications. Moreover, EV-encapsulated ncRNAs offer unique advantages over traditional cell-free ncRNAs due to their enhanced stability within the EVs, hence allowing for their detection in circulation for extended periods and enabling more sensitive and reliable biomarker measurements. Numerous studies have investigated the potential of EV-enclosed ncRNAs as biomarkers for CLD. MiRNAs, in particular, have gained significant attention due to their ability to rapidly respond to changes in cellular stress and inflammation, hallmarks of CLD pathogenesis. Elevated levels of specific miRNAs have been consistently associated with various CLD subtypes, including metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), and chronic hepatitis B and C. LncRNAs have also emerged as promising biomarkers for CLD. These transcripts are involved in a wide range of cellular processes, including liver regeneration, fibrosis, and cancer progression. Studies have shown that lncRNA expression profiles can distinguish between different CLD subtypes, providing valuable insights into disease progression and therapeutic response. Promising EV-enclosed ncRNA biomarkers for CLD included miR-122 (elevated levels of miR-122 are associated with MASLD progression and liver fibrosis), miR-21 (increased expression of miR-21 is linked to liver inflammation and fibrosis in CLD patients), miR-192 (elevated levels of miR-192 are associated with more advanced stages of CLD, including cirrhosis and HCC), LncRNA HOTAIR (increased HOTAIR expression is associated with MASLD progression and MASH development), and LncRNA H19 (dysregulation of H19 expression is linked to liver fibrosis and HCC progression). In the present review, we focus on the EV-enclosed ncRNAs as promising tools for the diagnosis and monitoring of CLD of various etiologies.

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

Naïve or Engineered Extracellular Vesicles from Different Cell Sources: Therapeutic Tools for Kidney Diseases.

Renal pathophysiology is a multifactorial process involving different kidney structures. Acute kidney injury (AKI) is a clinical condition characterized by tubular necrosis and glomerular hyperfiltration. The maladaptive repair after AKI predisposes to the onset of chronic kidney diseases (CKD). CKD is a progressive and irreversible loss of kidney function, characterized by fibrosis that could lead to end stage renal disease. In this review we provide a comprehensive overview of the most recent scientific publications analyzing the therapeutic potential of Extracellular Vesicles (EV)-based treatments in different animal models of AKI and CKD. EVs from multiple sources act as paracrine effectors involved in cell-cell communication with pro-generative and low immunogenic properties. They represent innovative and promising natural drug delivery vehicles used to treat experimental acute and chronic kidney diseases. Differently from synthetic systems, EVs can cross biological barriers and deliver biomolecules to the recipient cells inducing a physiological response. Moreover, new methods for improving the EVs as carriers have been introduced, such as the engineering of the cargo, the modification of the proteins on the external membrane, or the pre-conditioning of the cell of origin. The new nano-medicine approaches based on bioengineered EVs are an attempt to enhance their drug delivery capacity for potential clinical applications.

Human Liver Stem Cell Derived Extracellular Vesicles Alleviate Kidney Fibrosis by Interfering with the ß-Catenin Pathway through miR29b.

Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with ß-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The ß-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of ß-catenin was amplified both in vivo and in vitro, and ß-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that ß-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of ß-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which ß-catenin is down regulated by HLSC-EVs-induced miR29b expression.

Human Liver Stem Cells: A Liver-Derived Mesenchymal Stromal Cell-Like Population With Pro-regenerative Properties.

Human liver stem cells (HLSCs) were described for the first time in 2006 as a new stem cell population derived from healthy human livers. Like mesenchymal stromal cells, HLSCs exhibit multipotent and immunomodulatory properties. HLSCs can differentiate into several lineages under defined in vitro conditions, such as mature hepatocytes, osteocytes, endothelial cells, and islet-like cell organoids. Over the years, HLSCs have been shown to contribute to tissue repair and regeneration in different in vivo models, leading to more than five granted patents and over 15 peer reviewed scientific articles elucidating their potential therapeutic role in various experimental pathologies. In addition, HLSCs have recently completed a Phase 1 study evaluating their safety post intrahepatic injection in infants with inherited neonatal onset hyperammonemia. Even though a lot of progress has been made in understanding HLSCs over the past years, some important questions regarding the mechanisms of action remain to be elucidated. Among the mechanisms of interaction of HLSCs with their environment, a paracrine interface has emerged involving extracellular vesicles (EVs) as vehicles for transferring active biological materials. In our group, the EVs derived from HLSCs have been studied in vitro as well as in vivo. Our attention has mainly been focused on understanding the in vivo ability of HLSC-derived EVs as modulators of tissue regeneration, inflammation, fibrosis, and tumor growth. This review article aims to discuss in detail the role of HLSCs and HLSC-EVs in these processes and their possible future therapeutic applications.

Mesenchymal Stem Cell Derived Extracellular Vesicles Ameliorate Kidney Injury in Aristolochic Acid Nephropathy.

Limitations in the current therapeutic strategies for the prevention of progression of chronic kidney disease (CKD) to end stage renal disease has been a drawback to improving patient recovery. It is therefore imperative that a solution is found to alleviate this problem and improve the health and well-being of patients overall. Aristolochic acid (AA) induced nephropathy, a type of nephrotoxic CKD is characterised by cortical tubular injury, inflammation, leading to interstitial fibrosis. Extracellular vesicles derived from human bone marrow mesenchymal stem cells (MSC-EVs) display therapeutic properties in various disease models including kidney injury. In the current study, we intended to investigate the ability of MSC-EVs on ameliorating tubular injury and interstitial fibrosis in a mouse model of aristolochic acid nephropathy (AAN). The chronic model of AAN is comprised of an intraperitoneal injection of AA in NSG mice, followed by a three-day incubation period and then inoculation of MSC-EVs intravenously. This routine was performed on a weekly basis for four consecutive weeks, accompanied by the monitoring of body weight of all mice. Blood and tissue samples were collected post sacrifice. All animals administered with AA developed kidney injury and renal fibrosis. A gradual loss of body weight was observed, together with a deterioration in kidney function. Although no significant recovery was observed in weight loss following treatment with MSC-EVs, a significant reduction in: blood creatinine and blood urea nitrogen (BUN), tubular necrosis, and interstitial fibrosis was observed. In addition, infiltration of CD45 positive immune cells, fibroblasts, and pericytes which were elevated in the interstitium post AA induced injury, were also significantly reduced by MSC-EVs. Kidneys were also subjected to molecular analyses to evaluate the regulation of pro-fibrotic genes. MSC-EVs significantly reduced AA induction of the pro-fibrotic genes α-Sma, Tgfb1 and Col1a1. A downregulation in pro-fibrotic genes was also observed in fibroblasts activated by AA injured mTECs in vitro. Furthermore, meta-analyses of miRNAs downregulated by MSC-EVs, such as miR21, revealed the regulation of multiple pathways involved in kidney injury including fibrosis, inflammation, and apoptosis. These results therefore suggest that MSC-EVs could play a regenerative and anti-fibrotic role in AAN through the transfer of biologically active cargo that regulates the disease both at a protein and genetic level.

Human liver stem cells express UGT1A1 and improve phenotype of immunocompromised Crigler Najjar syndrome type I mice.

Crigler Najjar Syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1-/- mice, which closely mimic the pathological manifestations in CNSI patients. To assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture ex vivo, HLSC differentiated into hepatocyte-like cells showing UGT1A1 expression and activity. For the in vivo human cell engraftment and recovery experiments, DiI-labelled HLSC were injected into the liver of 5 days old NSG/Ugt1-/- pups which were analysed at postnatal Day 21. HLSC expressed UGT1A1 in vivo, induced a strong decrease in serum unconjugated bilirubin, thus significantly improving phenotype and survival compared to untreated controls. A striking recovery from brain damage was also observed in HLSC-injected mutant mice versus controls. Our proof-of-concept study shows that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1-/- mice, and show promises for the treatment of CNSI.

HLSC-Derived Extracellular Vesicles Attenuate Liver Fibrosis and Inflammation in a Murine Model of Non-alcoholic Steatohepatitis.

Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell-derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and, in particular, in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation at both morphological and molecular levels. In particular, we observed that, out of 29 fibrosis-associated genes upregulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function.

Human Liver-Derived Stem Cells Improve Fibrosis and Inflammation Associated with Nonalcoholic Steatohepatitis.

Cell therapy may be regarded as a feasible alternative to whole organ transplantation to treat end-stage liver diseases. Human liver stem cells (HLSCs) are a population of cells easily obtainable and expandable from a human adult liver biopsy. HLSCs share with mesenchymal stromal cells the same phenotype, gene expression profile, and differentiation capabilities. In addition, HLSCs show a specific commitment to the hepatic phenotype. Injection of HLSCs into immunodeficient mice fed with a methionine-choline-deficient diet to induce nonalcoholic steatohepatitis ameliorates liver function and morphology. In particular, HLSC treatment induced a reduction of liver fibrosis and inflammation at morphological and molecular levels. Moreover, HLSCs were able to persist for up to 3 weeks after the injection. In conclusion, HLSCs have healing effects in a model of chronic liver disease.

Mesenchymal Stromal Cell Derived Extracellular Vesicles Reduce Hypoxia-Ischaemia Induced Perinatal Brain Injury.

BACKGROUND: Neonatal hypoxic-ischemic (HI) insult is a leading cause of disability and death in newborns, with therapeutic hypothermia being the only currently available clinical intervention. Thus there is a great need for adjunct and novel treatments for enhanced or alternative post-HI neuroprotection. Extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have recently been shown to exhibit regenerative effects in various injury models. Here we present findings showing neuroprotective effects of MSC-derived EVs in the Rice-Vannucci model of severe HI-induced neonatal brain insult. METHODS: Mesenchymal stromal/stem cell-derived EVs were applied intranasally immediately post HI-insult and behavioral outcomes were observed 48 h following MSC-EV treatment, as assessed by negative geotaxis. Brains were thereafter excised and assessed for changes in glial responses, cell death, and neuronal loss as markers of damage at 48 h post HI-insult. RESULTS: Brains of the MSC-EV treated group showed a significant decrease in microglial activation, cell death, and percentage tissue volume loss in multiple brain regions, compared to the control-treated groups. Furthermore, negative geotaxis test showed improved behavioral outcomes at 48 h following MSC-EV treatment. CONCLUSION: Our findings highlight the clinical potential of using MSC-derived EVs following neonatal hypoxia-ischaemia.

Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis.

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.

Human Liver Stem Cell-Derived Extracellular Vesicles Prevent Aristolochic Acid-Induced Kidney Fibrosis.

With limited therapeutic intervention in preventing the progression to end-stage renal disease, chronic kidney disease (CKD) remains a global health-care burden. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by inflammation, tubular injury, and interstitial fibrosis. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) have been reported to exhibit therapeutic properties in various disease models including acute kidney injury. In the present study, we aimed to investigate the effects of HLSC-EVs on tubular regeneration and interstitial fibrosis in an AA-induced mouse model of CKD. NSG mice were injected with HLSC-EVs 3 days after administering AA on a weekly basis for 4 weeks. Mice injected with AA significantly lost weight over the 4-week period. Deterioration in kidney function was also observed. Histology was performed to evaluate tubular necrosis, interstitial fibrosis, as well as infiltration of inflammatory cells/fibroblasts. Kidneys were also subjected to gene array analyses to evaluate regulation of microRNAs (miRNAs) and pro-fibrotic genes. The effect of HLSC-EVs was also tested in vitro to assess pro-fibrotic gene regulation in fibroblasts cocultured with AA pretreated tubular epithelial cells. Histological analyses showed that treatment with HLSC-EVs significantly reduced tubular necrosis, interstitial fibrosis, infiltration of CD45 cells and fibroblasts, which were all elevated during AA induced injury. At a molecular level, HLSC-EVs significantly inhibited the upregulation of the pro-fibrotic genes α-Sma, Tgfb1, and Col1a1 in vivo and in vitro. Fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in AA injured mice. Treatment with HLSC-EVs downregulated 14 pro-fibrotic genes in total, out of which, 5 were upregulated in mice injured with AA. Analyses of the total mouse miRnome identified several miRNAs involved in the regulation of fibrotic pathways, which were found to be modulated post-treatment with HLSC-EVs. These results indicate that HLSC-EVs play a regenerative role in CKD possibly through the regulation of genes and miRNAs that are activated during the progression of the disease.

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

BACKGROUND: Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. METHODS: HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. RESULTS: Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme and mRNA. In fact, EVs from ASS1-knockdown HLSCs contained low amounts of ASS1 mRNA and protein, and were unable to restore urea production in hepatocytes differentiated from ASS1-HLSCs. CONCLUSIONS: Collectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.

Recellularization of rat liver scaffolds by human liver stem cells.

In the present study, rat liver acellular scaffolds were used as biological support to guide the differentiation of human liver stem-like cells (HLSC) to hepatocytes. Once recellularized, the scaffolds were maintained for 21 days in different culture conditions to evaluate hepatocyte differentiation. HLSC lost the embryonic markers (alpha-fetoprotein, nestin, nanog, sox2, Musashi1, Oct 3/4, and pax2), increased the expression of albumin, and acquired the expression of lactate dehydrogenase and three subtypes of cytochrome P450. The presence of urea nitrogen in the culture medium confirmed their metabolic activity. In addition, cells attached to tubular remnant matrix structures expressed cytokeratin 19, CD31, and vimentin. The rat extracellular matrix (ECM) provides not only a favorable environment for differentiation of HLSC in functional hepatocytes (hepatocyte like) but also promoted the generation of some epithelial-like and endothelial-like cells. When fibroblast growth factor-epidermal growth factor or HLSC-derived conditioned medium was added to the perfusate, an improvement of survival rate was observed. The conditioned medium from HLSC potentiated also the metabolic activity of hepatocyte-like cells repopulating the acellular liver. In conclusion, HLSC have the potential, in association with the natural ECM, to generate in vitro a functional "humanized liver-like tissue."

Human liver stem cells and derived extracellular vesicles improve recovery in a murine model of acute kidney injury.

INTRODUCTION: Several cellular sources of stem cells have been tested in the attempt to yield innovative interventions in acute kidney injury (AKI). Human liver stem cells (HLSCs) are cells isolated from the normal adult human liver which are gaining attention for their therapeutic potential. In the present study, we investigated whether HLSCs and the derived extracellular vesicles may promote tubular regeneration after AKI induced by glycerol injection in severe-combined immune-deficient mice. METHODS: HLSCs were expanded and conditioned medium (CM) and extracellular vesicles (EVs) were purified. HLSCs and their bioproducts were tested in a model of AKI induced by intra-muscle glycerol injection. Renal function and morphology were evaluated five days after induction of damage. The effect of EVs on proliferation and apoptosis of murine renal tubular cells was tested in vitro. RESULTS: We found that intravenous injection of 3.5×105 HLSCs into mice three days after induction of AKI significantly improved functional and morphological recovery. The injection of HLSCs decreased creatinine and urea, as well as hyaline cast formation, tubular necrosis and enhanced in vivo tubular cell proliferation. The effect of soluble factors release by HLSCs in the regenerative processes was also studied. CM produced by HLSCs, mimicked the effect of the cells. However, depletion of EVs significantly reduced the functional and morphological recovery of CM. Moreover, we found that purified HLSC-derived EVs ameliorated renal function and morphology in a manner comparable to the cells. In vitro HLSC-derived EVs were shown to stimulate proliferation and inhibit apoptosis of murine renal tubular cells. CONCLUSIONS: These results indicate that HLSCs increase recovery after AKI. EVs are the main component of HLSC-derived CM capable of promoting regeneration in experimental AKI.