Fig. 6

Urea production and ASS1 enzymatic activity. a Histogram representing the urea production ability of hepatocytes differentiated under RCCS conditions for 4 days from human liver stem cells (HLSCs), HLSCs from the liver of a patient suffering from citrullinemia type 1 (ASS1-HLSCs), ASS1-HLSCs treated with 1 × 10¹⁰ HLSC-derived extracellular vesicles (EVs), ASS1-HLSCs treated with 1 × 10¹⁰ HLSC-EVs from ASS1 shRNA transfected HLSCs (shRNA EVs) or with 1 × 10¹⁰ EVs derived from human fibroblasts (hFib EV). Data are expressed as mean ± SD of four different experiments. ANOVA with Newmann-Keuls multicomparison tests was performed. *p < 0.05, ASS1-HLSC vs HLSC; ^# p < 0.05, ASS1-HLSC + HLSC-EVs vs ASS1-HLSC; ^§ p < 0.05, ASS1-HLSC + shRNA EVs vs ASS1-HLSC + HLSC-EVs; ^{^} p < 0.05 ASS1-HLSC + hFib EVs vs AS-HLSC + EVs. b The enzymatic activity of ASS1 immunoprecipitated from either rat liver extract (RLE; positive control), hepatocytes differentiated in RCCS for 4 days from HLSCs, ASS1-HLSCs, or ASS1-HLSCs treated with 1 × 10¹⁰ HLSC-EVs were measured. For selected experiments the enzyme activity was measured in the presence of α-methyl-dl-aspartic acid (MDLA), a specific inhibitor of ASS1 enzymatic activity, and using ASS1-HLSCs previously transfected with ASS-siRNA. Data are expressed as mean ± SD of four separate experiments. ANOVA with Newmann-Keuls multicomparison tests was performed. *p < 0.05, HLSC + MDLA vs HLSC; ^§ p < 0.05, ASS1-HLSC vs HLSC; ^# p < 0.05, ASS1- HLSC + EVs vs ASS1-HLSC; ^{^} p < 0.05, ASS1- HLSC + EV/MDLA vs ASS1-HLSC + EV; °p < 0.05, ASS1-HLSC + siRNA/EV vs ASS1-HLSC + EV