The primary objective of the study is to generate and characterize three-dimensional models, called "assembloids", composed of the main liver cell populations (in particular from the co-culture of organoids with stellate cells, responsible for fibrogenesis, deriving from clinical samples). These models will be used in order to imitate the first phases of the onset of steatohepatitis, in conditions of altered lipid metabolism (induced through exposure to the main environmental determinants of this condition: excess fatty acids, fructose, cholesterol) in the presence or absence of the mutation I148M of PNPLA3. Other genetic variants will also be analyzed, such as TM6SF2, MBOAT7 and GCKR, which have previously been correlated with the development of non-alcoholic steatohepatitis.
Further objectives will be: 1) identify new biomarkers of pathological activation of human stellate cells and progression of liver damage, to be subsequently validated in clinical case series for future use in clinical management for individual risk stratification; 2) study the epigenetic factors that underlie the onset of non-alcoholic steatohepatitis and its progression to fibrosis, cirrhosis and HCC; 3) evaluate the impact of antisense oligonucleotides directed against PNPLA3 on the severity of the "steatohepatitic" phenotype (lipid accumulation, lipotoxicity and inflammation and fibrogenesis) in assembloids
liver biopsy for suspected non-alcoholic steatohepatitis (NASH) at the time of diagnosis;
liver resection for hepatocarcinoma or other liver lesions (including secondaries from other neoplasms and benign focal lesions, which will allow obtaining healthy starting liver tissue);
post-transplant healthy liver biopsies;
cholecystectomies.
None
Single group
Liver resection to isolate cells
GeneticIsolation and culture of organoids Isolation of hepatic stellate cells Isolation and culture of sinusoidal stellate cells Generation of assembloids
Generation of treatment-related assembloids
Isolation of epithelial cells to impact assessment of the I148M variant on the steatohepatic phenotype in human liver assembloids and analysis of the variants of the TM6SF2, MBOAT7 and GCKR genes previously related to the development of non-alcoholic steatohepatitis
Rate of PNPLA3 p.I148M variant in steatohepatitis development through human liver organoids
* Knowledge of the gene expression of lipotoxicity and expression of inflammatory markers; * Knowledge of the molecular mechanisms to evaluate the state of activation of star cells, the metabolism of retinol and the deposition of extracellular matrix and estimate
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