Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency

Maria Beatriz Herrera Sanchez,Valentina Fonsato,Maria Chiara Deregibus,Emanuela Tolosano,Rossana Critelli,Giovanni Camussi,Sara Petrillo,Sharad Kholia,Ciro Tetta,Marco Spada,Renato Romagnoli,Mauro Salizzoni,Sara Previdi,Stefania Bruno

doi:10.1186/s13287-017-0628-9

Abstract

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

Highlights

Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate
We investigated whether Extracellular vesicle (EV) derived from normal Human liver stem cell (HLSC) had the ability to carry and transfer wild-type ASS1 to mature hepatocytes differentiated from ASS1-mutated HLSCs, thereby restoring enzyme activity and urea production
Characterization and in vitro differentiation of citrullinemia type I-derived ASS1-HLSCs ASS1-HLSCs derived from the liver of a patient with citrullinemia type I were characterized to confirm their expression of stem cell markers similar to HLSCs

Summary

Introduction

Argininosuccinate synthase (ASS) 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; due to limited organ availability, substitute therapies are required. 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. Citrullinemia type I is an autosomal recessive disorder caused by a deficiency in argininosuccinate synthase (ASS; common gene symbol alias: ASS1), an enzyme that catalyzes the third reaction of the urea cycle [1]. Due to the limited availability of donor livers, alternative treatments such as gene therapy are currently under investigation [2]

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