Ciro Tetta

A robust and easy-to-use tool for the ex vivo dynamic evaluation of pancreatic islet (PI) function is essential for further development of novel cell-based therapeutic approaches to treating diabetes. Here, we developed four different glucose perifusion protocols (GPPs) in a microfluidic perifusion system (MPS), based entirely on commercially available components. After validation, the GPPs were used to evaluate C-peptide secretion profiles of PIs derived from different donors (healthy, obese, and type 2 diabetic) and from human liver stem-cell-derived islet-like structures (HLSC-ILS). Using this device, we demonstrated that PIs derived from healthy donors displayed a physiological C-peptide secretion profile as characterized by the response to (a) different glucose concentrations, (b) consecutive pulses of high-glucose concentrations, (c) a glucose threshold ranging from 5–8 mM, and (d) a constant high-glucose perifusion in a biphasic manner. Moreover, we were able to detect a dysregulated secretion profile in PIs derived from both obese and type 2 diabetes mellitus (T2DM) donors. Finally, we also evaluated the kinetic secretion profiles of HLSC-ILS, demonstrating that, nonetheless, with a lower amplitude of secretion compared to PI derived from healthy donors, they were already glucose-responsive on day seven post-differentiation. In conclusion, we have provided evidence that our MPS is a versatile device and may represent a valuable tool to study insulin-producing cells in vitro.

EFFECTS OF EXTRACELLULAR VESICLES IN REPAIR OF ACUTE AND CHRONIC RENAL INJURY EFFECTS OF EXTRACELLULAR VESICLES IN REPAIR OF ACUTE AND CHRONIC RENAL INJURY

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. 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. 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. Collectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.

Introduction and Aims: Acute Kidney Injury (AKI) requiring Renal Replacement Therapy (RRT) occurs in 5 to 6% of critically ill patients and is associated with high mortality and significant health resource utilization.

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.

Optimizing anemia treatment in hemodialysis (HD) patients remains a priority worldwide as it has significant health and financial implications. Our aim was to evaluate in a large cohort of chronic HD patients in Fresenius Medical Care centers in Spain the value of cumulative iron (Fe) dose monitoring for the management of iron therapy in erythropoiesis-stimulating agent (ESA)-treated patients, and the relationship between cumulative iron dose and risk of hospitalization.

While fluid overload (FO) and alterations in the autonomic nervous system (ANS) such as hypersympathetic activity, are known risk factors for cardiovascular morbidity and mortality in patients on chronic hemodialysis (HD), their relationship has not been thoroughly studied. In this observational study involving 69 patients on chronic HD, FO was assessed by whole body bioimpedance measurements before the midweek HD session and ANS activity reflected by Heart Rate Variability (HRV) was measured using 24-hour Holter electrocardiogram recordings starting before the same HD treatment. In total, 13 different HRV indices were analyzed, comprising a mixture of time domain, frequency domain and complexity parameters. A correlation analysis was performed between the HRV indices and hydration status indices. Successively, patients were retrospectively assigned to a high FO (H, FO > 2.5 L) or low FO (L, FO ≤ 2.5 L) group and these were further compared also after stratification by diabetes mellitus. Finally, a small number of patients without diabetes with significant and persistent FO were followed up for 3 months post-study to investigate how normalization of fluid status affects HRV. SDANN, VLF, LZC and HF% parameters significantly correlate with FO (correlation coefficients were respectively r = –0.40, r = –0.37, r = –0.28 and r = 0.26, p-value < 0.05). Furthermore, LF% and LF/HF were inversely correlated with hydration status (correlation coefficients were respectively r = –0.31 and r = -0.33, p-value < 0.05). These results indicate an association between FO and reduced HRV, higher parasympathetic activation and reduced sympathetic response to the HD session. Indeed, group H tended to have lower values of SDANN, VLF and LZC, and higher values of HF% than patients in the L group. Finally, there was a trend towards lower LF% measured during the last 30 minutes of HD for the H group versus the L group. Reduction in FO achieved over 3 months by implementation of a strict fluid management plan resulted in an increase of HRV. Our results suggest that depressed HRV is associated with fluid overload and that normalization of hydration status is accompanied by improved HRV.

There has been increasing interest in the application of mesenchymal stem cells (MSCs) in regenerative medicine in recent years. In this context, the beneficial effects of MSCs have been ascribed mainly to a paracrine action rather than to direct replacement of the injured tissue. Indeed, MSCs produce a great variety of trophic and immunomodulatory factors. In this chapter, we provide an overview of growth factors and chemokines involved in stimulation of cell proliferation, inhibition of apoptosis, enhancement of angiogenesis, and suppression of inflammatory and immune response. In addition, we discuss the emerging role of the extracellular vesicles released from MSCs as possible paracrine mediators.

Observational study of patients on hemodialysis (HD) in FMC® Spain clinics over the years 2009 and 2010. The data were collected from the EuClid® database, implemented in the clinics of FMC®, which complies with the following feature: record online, compulsory, conducted in patients incidents and that it covers the entire population on HD in these clinics. Its aim is to understand the characteristics of patients and treatment patterns, comparing them with other studies described in the literature and in order to improve their prognosis and quality of life. Include 2637 incidents patients and 4679 prevalent, which makes a total of 7316 patients. In prevalent patients: 24.4% were diabetic; 76.3% had cardio-vascular disease (CVD) and 13.4% cancer. Among the incidents, these percentages were: 33.5% diabetic; 80.6% had CVD and 12.6% cancer. The prevalent patients had such as vascular access: FAV 68.5%, prosthesis 5.6%, permanent catheter 23.7% and 2.3% temporary catheter. The average of the duration of the sessions of HD was 230 minutes. 23.2% of the prevalent patients were on on-line hemodiafiltration. These patients hospitalization rates were 0.46 hospitalizations per incident patient per year and 0.52 per prevalent patient per year. The annual gross mortality rate was 12%. The mortality of the patients in this study HD is smaller than these of the Spanish Registry of Dialysis and Transplant (GRER). The result of morbidity and mortality of the FMC clinics of Spain can, therefore, be as good compared with these of the GRER and other international series. That does not mean that there are not areas of improvement as the increase in the time of dialysis, the percentage of patients on on-line hemodiafiltration convective techniques and the percentage of FAV.