Human Liver ORganoids as a Model to Study the Role of the I148M Variant of the PNPLA3 Gene in the Development of Non-Alcoholic SteatOhepatitis (NASH)
Phase: NA
Conditions: NAFLD
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

<Human Liver Organoids as a Model to Study the
role of the I148M variant of the genePNPLA3in the 
development of Non-Alcoholic Steatohepatitis (NASH)=
Acronym: REASON
Protocol version number: v. 2.0
Date: January 24, 2024
Promoter:IRCCS Ca' Granda Foundation Maggiore Hospital Polyclinic, Via Sforza 28, 20122
Milan, Italy
Coordinating center:UOC Transfusion Center
IRCCS Ca' Granda Foundation Maggiore Hospital Polyclinic, Via Sforza 35, 
20122 Milan, Italy
Principal Investigator:Prof. Luca Vittorio Valenti
PRIVACY STATEMENT
All information presented in this document will be considered confidential and will remain the exclusive property of 
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. The use of such confidential information must be limited to 
the recipient for the agreed purpose and must not be disclosed, published or otherwise communicated to unauthorized 
persons, for any reason, in any form without the prior written consent of Fondazione IRCCS Ca' Granda Ospedale 
Maggiore Policlinico.
Translated from Italian to English - www.onlinedoctranslator.com

FLOWCHARTS
Screening
Intervention
Period
(-t1)
(t1)
Enlistment
informed consent
-
inclusion/exclusion criteria
-
Administration of the intervention
Biological sample collection
-
LIST OF ABBREVIATIONS
ADH: alcohol dehydrogenase ARAT: 
retinol acyltransferase GWAS: genome-
wide association study HSCs: Hepatic 
stellate cells
LRAT: lecithin retinol acyl-transferase 
MMP13: matrix metalloprotease 13 
NAFLD: nonalcoholic fatty liver disease 
NASH: nonalcoholic steatohepatitis
PNPLA3: Patatin-like phospholipase domain-containing protein 3 
RA: retinoic acid
RALDH: retinaldehydedehydrogenase-- TGF³1: 
transforming growth factor ³1 TIMP: tissue 
inhibitor matrix metalloproteases
-SMA: ³-smooth muscle actin

TITLE PAGE
FLOWCHART
LIST OF ABBREVIATIONS INDEX 
1.INTRODUCTION
1.1 Background and rationale
2.OBJECTIVE OF THE EXPERIMENTATION
2.1 Primary objective
2.2 Secondary objective(s)
3. DESIGN OF THE STUDIO
3.1 Study design
3.2 Inclusion criteria
3.3 Exclusion criteria
4.PROCEDURES RELATING TO THE STUDY
4.1 Intervention
4.2 Isolation and culture of organoids
4.3 Isolation and culture of stellate cells
4.4 Generation of assemblyloids
4.5 In vitro treatment
4.6 Genetic analysis
4.7 RNA isolation
4.8 Characterization of the activation state of human hepatic stellate cells
5.ENDPOINT
5.1 Primary Endpoint
5.2 Secondary Endpoints
6. DURATION/TIMELINE OF THE STUDY
7.STATISTICAL ANALYSIS

7.1 Sample size and data analysis
8.ADVERSE EVENTS
9.RISK/BENEFIT ASSESSMENT
10. STUDIO MANAGEMENT
10.1 Data collection and management
10.2 Regulatory aspects and ethical considerations
10.2.1 Approval by the Competent Authority
10.2.2 Ethics Committee Approval
10.2.3 Informed consent
10.3 Duties of the experimenter
10.4 Study monitoring
10.5 Quality assurance of the study
10.6 Closing of the study
10.7 Document storage
10.8 Disclosure of information regarding scientific discovery
10.8.1 Confidentiality
10.8.2 Publications
10.9 Intellectual property rights on the results of the study
11. Indemnity and compensation in case of damages
12.Financial agreements
13.Disclosure on conflicts of interest
14.References

1. INTRODUCTION
1.1 Background and rationale
Nonalcoholic fatty liver disease (NAFLD), defined as the accumulation of fat in the liver not 
explained by risky alcohol consumption, is the leading cause of liver damage and occurs in 
approximately one-third of the population (25330%). NAFLD encompasses a broad spectrum of 
conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). NASH is 
defined histologically by the coexistence of steatosis, lobular inflammation, and hepatocellular 
damage and is frequently complicated by the development of fibrosis. NASH occurs in 20330% 
of patients with NAFLD and may progress to cirrhosis and hepatocellular carcinoma (HCC) (1). 
Recent advances in genetics have highlighted that genetic factors play a key role in the 
susceptibility, severity, and long-term prognosis of the disease (2). In particular, in a genome-
wide association study (GWAS) conducted on a multiethnic population, the gene was identified
PNPLA3 (Patatin-likephospholipase domain-containingprotein 3),located on chromosome 22, as 
the major genetic factor that strongly associates with intrahepatic fat content, independently of 
body mass and insulin resistance (3). In particular, the polymorphism in the genePNPLA3 (
rs738409, C>G), which determines an amino acid substitution from isoleucine to methionine in 
position 148 of the protein (I148M), currently represents the most important genetic predictor 
of steatosis, steatohepatitis and progression of liver damage and strongly increases the risk of 
developing hepatocellular carcinoma (4, 5).PNPLA3encodes a protein, also called adiponutrin, 
located at the level of the endoplasmic reticulum and on the surface of lipid droplets, with an 
important enzymatic function (triacylglycerol lipase), involved in the hydrolysis of triglycerides in 
hepatocytes and adipocytes and in the catabolism of retinol esters in stellate cells. The presence 
of the amino acid methionine in position 148 would lead to a loss of function of the protein with 
accumulation at the surface of lipid droplets and reduced remodeling of triglycerides (6). 
Furthermore, it has been demonstrated that PNPLA3, following induction by TGF--, regulates the 
metabolism of retinol in stellate cells (retinyl-esterase activity) determining the release of retinol
ex vivoandin vitro (7). The risk variant 148M instead seems to prevent the release of retinol. 
Furthermore, it has been observed that the progression of chronic liver diseases related to 
steatosis is associated with cellular senescence and therefore patients carrying the risk variant 
(148M) show an up-regulation of cellular senescence andpathwaysfibrogenic.
These preclinical studies suggestPNPLA3as a new therapeutic target, especially with regard to the 
possibility of reducing its expression in the mutated variant. Therefore, screening based on the 
determination of the I148M variant status ofPNPLA3could be fundamental for designing future precision 
medicine approaches. Although the I148M variant ofPNPLA3causes progression of liver damage to 
advanced fibrosis and hepatocellular carcinoma (1), the phenotypic expression is highly variable and 
depends on the coexistence of other genetic factors and environmental factors. Furthermore, the relative 
contribution of the I148M variant carrier state on the steatohepatitis phenotype in

hepatocytes vs. stellate cells is not yet clear. The study of new molecular pharmacological 
approaches that aim at regulating the expression and activity of PNPLA3 for the prevention and 
treatment of liver diseases is however still difficult, due to both the limitations of experimental 
models in animals (cost, suffering, different regulation of lipid metabolism and PNPLA3 compared to 
humans) and the difficulty of reproducing NASH in modelsin vitro.
The current modelsin vitro,based on liver cell lines, are in fact unable to reproduce the intrinsic 
cellular heterogeneity, the three-dimensional architecture of the tissue and the cell-cell interactions. 
These characteristics are fundamental in the study of the processes that can lead to the onset of 
steatosis, progressing to a fibrotic, cirrhotic and tumorous state following an external insult. 
Organoids, three-dimensional (3D) cellular structures, generated both from induced pluripotent 
stem cells and from tissue-resident adult stem cells, are recently establishing themselves as a new 
culture model to overcome the limitations of traditional cell culture systems, emerging as powerful 
tools for the study of human diseases (8, 9). This is possible because they are able to stably preserve 
the genetic information of the autologous tissue and mimic the physiological and pathological state 
of the tissue of interest. Despite the cellular heterogeneity exhibited by organoids, these structures 
present some limitations such as the low capacity to mimic the architecture of the mature organ of 
interest due to the absence of cells that compose the surrounding environment (10, 11). This 
limitation can be overcome by creating 3D cultures, defined as <assembloids=, composed of the cells 
that constitute the organoids in co-culture with cells of the surrounding environment (stromal, 
stellate, endothelial cells, etc.), capable of recreating a more precise architecture of the starting 
tissue, represented by the epithelial component surrounded by its stroma (12, 13). These structures 
will be the basis for studying the molecular mechanisms that underlie the onset of steatosis and its 
progression to a fibrotic condition, due to the activation of stellate cells, following the damage 
suffered by hepatocytes.
2. OBJECTIVE OF THE EXPERIMENT
The aim of this project is to study the cellular and molecular mechanisms through which the variant 
at risk ofPNPLA3 (148M) accelerates the progression of liver damage in models based on the 
development of liver organoids and assemblyloids, and to test in these the efficacy of new molecular 
pharmacological approaches aimed at silencing the expression of the mutated protein variant in 
improving the pathological phenotype. Our hypothesis is that the loss of function ofPNPLA3in 
hepatocytes and hepatic stellate cells may represent a link between accumulation of hepatic fat, 
development of lipotoxicity and inflammation, fibrosis and finally liver cancer. The 148M variant 
could determine a persistent and uncontrolled activation of stellate cells (HSCs) due to an altered 
ability to release retinol. Activated HSCs would then undergo premature senescence with 
consequent release of pro-inflammatory and carcinogenic mediators. Silencing of PNPLA3 by using 
antisense RNA would therefore lead to a greater improvement of the

hepatocellular damage specifically in models derived from cells carrying the risk variant.
2.1 Primary objective
The primary objective of the study conducted at the Foundation is to generate and characterize 
three-dimensional models, called <assembloids=, composed of the main liver cell populations (in 
particular from the coculture of organoids derived from clinical samples together with stellate 
cells responsible for fibrogenesis). These models will be used to mimic the early stages of 
steatohepatitis onset in conditions of altered lipid metabolism (induced by exposure to the main 
environmental determinants of this condition: excess fatty acids, fructose, cholesterol) in the 
presence or absence of the I148M mutation ofPNPLA3. Other genetic variants will also be 
evaluated.
2.2 Secondary objectives
1. Identification of novel non-invasive biomarkers of pathological activation of human stellate 
cells and progression of liver damage to be subsequently validated in clinical series for 
future use in clinical management for individual risk stratification.
2. Study of epigenetic factors underlying the onset of non-alcoholic steatohepatitis and its 
progression to fibrosis, cirrhosis and HCC.
3. Evaluation of the impact of antisense oligonucleotides directed against PNPLA3 on the severity of the 
<steatohepatitic= phenotype (lipid accumulation, lipotoxicity and inflammation and fibrogenesis) in 
assembloids.
3. STUDY DESIGN
3.1 Study design
Genetic interventional, with the collection of biological, non-pharmacological, monocentric material.

RESPONSIBILITY (role of the promoter and collaborators)
Operational unit
Participant Name
Role and functions in the study
Patient recruitment and sample 
characterization; Isolation and 
generation of organoids, spheroids 
and tissues;
Characterization of organoids by 
gene and protein expression Data 
analysis
Department of Transfusion 
Medicine and Hematology, 
Fondazione IRCCS Ca' Granda 
Ospedale Maggiore Policlinico
Dr. Daniele Prati
Prof. Luca Valenti (principal 
investigator)
Internal collaborations:
Operational unit
Participant Name
Role and functions in the study
UOC General Surgery and Liver 
Transplants,
IRCCS Foundation Ca' Granda 
Hospital Maggiore Polyclinic
Professor Giorgio Rossi,
Dr. Daniele Dondossola
Reporting of eligible patients and 
characterization of samples
Scientific Direction
IRCCS Foundation Ca' Granda 
Hospital Maggiore Polyclinic
Dr. Stefano Gatti
Sample characterization and 
support in cell isolation protocols
External body that will handle the analysis of the biological sample:
Operational unit
Participant Name
Role and functions in the study
Institute for Liver and Digestive 
Health, Division of Medicine, 
University College London
Prof. Krista Rombouts
Immunohistochemical and biomarker 
analyses on liver histological samples
3.2 Inclusion criteria
Patients of legal age who have given consent to participate in the study will be included and will be listed for the 
following procedures:
- liver biopsy for suspected nonalcoholic steatohepatitis (NASH) at the time of diagnosis;
- liver resection for hepatocellular carcinoma or other liver lesions (including secondary lesions from 
other neoplasms and benign focal lesions, which will allow obtaining starting liver tissue

healthy);
- post-transplant healthy liver biopsies;
- cholecystectomies.
You will also be required:
- willingness to sign informed consent for the study
- availability of DNA sample for genetic analysis and clinical data,
- blood sampling for genetic and epigenetic analysis and analysis of non-coding RNAs 
(lncRNAs, miRNAs and circRNAs).
3.3 Exclusion criteria
Patients with the following will be excluded:
- positivity for chronic viral hepatitis (HCV-RNA and/or HBsAg);
- positivity to other liver diseases such as autoimmune and viral hepatitis (hepatitis B and C), 
hereditary hemochromatosis, alpha-1-antitrypsin deficiency, Wilson's disease.
4. PROCEDURES RELATING TO THE STUDY
4.1 Intervention
In collaboration with the Liver Transplant Unit of the Foundation, samples of discarded tissue from 
patients undergoing liver resection will be collected (both intra-tumoral and extra-tumoral tissues in the 
case of hepatocarcinoma) or taken from the whole liver post-transplant. This procedure will not entail any 
additional risk for the patient compared to the usual routine, nor will it reduce the availability of material 
for standard pathological analyses.
These samples will allow us to isolate cells (ovalocytes 3 liver progenitor cells) to generate 
organoids from normal liver tissue (cholecystectomy) and suspected NASH.
We plan to collect up to 20 independent samples per tissue type. The 
groups considered will be:
- organoids, cultures of sinusoidal stellate cells isolated from 20 patients with CC genotype (I148I)
- organoids, cultures of sinusoidal stellate cells isolated from 20 patients with CG genotype 
(I148M)
- organoids, cultures of sinusoidal stellate cells isolated from 20 patients with GG genotype

(M148M)
A blood sample (approximately 7 cc) will also be taken, in addition to those already 
scheduled during regular clinical checks, at the time of surgery or liver biopsy.
It is planned to share a portion of the liver biopsy samples for immunohistochemical analysis to be 
carried out at the Division of Medicine, Institute for Liver and Digestive Health, University College London, 
in collaboration with Prof. Krista Rombouts, in order to further evaluate the expression of PNPLA3 
associated with liver disease, subject to the preparation of an appropriate MTA.
4.2 Isolation and culture of organoids
Biopsy fragments or fragments obtained from surgical resections will be transported in Celsior 
perfusion solution at 4°C and will be processed at the Translational Medicine laboratory, UOC 
Transfusion Center of the Foundation within 24 hours of collection to isolate liver organoids. The 
tissues will be mechanically fragmented into small pieces avoiding reducing them to single cells; this 
will increase the efficiency of organoid formation. Then the fragments will be further subjected to 
enzymatic digestion with a solution containing Collagenase and DNase at 37°C. The cell clusters 
obtained from enzymatic digestion will be included inreducedgrowthfactorMatrigel. Once the 
Matrigel has polymerized, the complete culture medium for liver organoids will be added: Advanced 
DMEM/F12 supplemented with 1% N2 and 1% B27 (both from GIBCO), 1.25 mM N-Acetylcysteine   
(Sigma), 10 nM Leu-Gastrin (Sigma), 50 ng/ml EGF (Peprotech), 1 -g/ml RSPO1 (Peprotech), 100 ng/ml 
FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 10 mMNicotinamide (Sigma), 5 -M A83.01 (Peprotech) 
and 10 -M Forskolin (Peprotech). In order to increase the efficiency of organoid isolation, 25 ng/ml 
Noggin (Peprotech), 100 ng/ml Wnt3a (peprotech) and 10 -M Rock inhibitor Y27632 (Peprotech) will 
be added to the medium for the first few days. The organoids will then be differentiated towards a 
hepatocyte phenotype.
4.3 Isolation and culture of sinusoidal stellate cells
A part of the biopsy fragments or those obtained from surgical resections, once digested to obtain 
the epithelial cells that will form the organoids, will be processed to obtain hepatic stellate cells (HSC) 
and sinusoidal cells (LSEC). The latter will be isolated by exploiting their low density, due to the 
presence of lipid droplets that characterize them. The stellate cells will be cultured on plastic and we 
will characterize their activation state and the purity of the cell population, by flow cytometry and 
quantitative Real Time PCR (RT-qPCR), evaluating the presence of the

retinol and specific cellular markers (15). Sinusoidal cells will be cultured on plastic pre-
treated with a fibronectin coating and grown in the presence of the growth factor VEGF.
4.4 Generation of assemblyloids
In order to obtain the assemblyloids, both the organoids and the stellate and sinusoidal cells will be 
dissociated into single cells by enzymatic digestion using trypsin. The cells will then be included in
reducedgrowthfactorMatrigel in different ratios in order to mimic the physiological conditions 
present in the liver tissue. Once the Matrigel has polymerized, the culture medium appropriately 
formulated to allow the survival of both hepatocytes, ductal cells and stellate cells will be added: 
Advanced DMEM/F12 supplemented with 1% N2 and 1% B27 (both from GIBCO), 1.25 mM N-
Acetylcysteine   (Sigma), 10 nM Leu-Gastrin (Sigma), 50 ng/ml EGF (Peprotech), 25 ng/ml HGF 
(Peprotech) and 2% FBS (GIBCO).
4.5 Treatmentin vitro
It has recently been shown that the addition of oleate and palmitate to the culture medium is 
able to increase the formation of lipid droplets in hepatocytes and primary stellate cells, 
simultaneously causing a down-regulation of the expression ofPNPLA3.Conversely, in the 
absence of retinol and palmitate, a reduction in intracellular lipid content and an increase in the 
expression ofPNPLA3 (7).
The generated assembloids will be treated for 36 or 120 hours with oleic acid and palmitic acid (300 
µM both) (Sigma Aldrich, St Louis, MO). Treatments with the addition of cholesterol, fructose, insulin 
and low concentrations of ethyl alcohol will also be tested to mimic a condition of dysmetabolism, 
unbalanced diet and the main environmental determinants of liver disease. This experiment will 
allow us to evaluate differences in the accumulation and subsequent release of lipid droplets in cells 
with different genotypes ofPNPLA3.After 36 hours, the lipid droplet content will be visualized by 
OilRed O histochemical staining. An analysis of stellate cell activation will also be performed after 36 
hours. Instead, after 120 hours, the assembloids will be partly fixed in formaldehyde to analyze 
morphology, biomarkers and fibrotic status and partly frozen in liquid nitrogen for further gene 
expression analysis.
PNPLA3 will be silenced using antisense RNA (16)
4.6 Genetic Analysis
DNA will be extracted by phenol-chloroform from peripheral blood and the I148M variant

PNPLA3(rs738409) will be determined by TaqMan assay on ABI 7500 Fast apparatus (Life 
Technologies, Carlsbad, CA, USA) (17).
4.7 RNA isolation
RNA will be extracted from cultured organoids using Trizol reagent (Life Technologies, Carlsbad, CA), 
according to the manufacturer's instructions. 1 µg of total RNA will be reverse transcribed using the 
VILO kitrandom hexamerssynthesissystem (Life Technologies, Carlsbad, CA). Gene expression will be 
assessed by quantitative real-time PCR (RT-qPCR) using an ABI 7500 Fast thermal cycler (Life 
Technologies, Carlsbad, CA) and SYBR Green chemistry (Fast SYBR Green Master Mix; Life 
Technologies, CA). All reactions will be performed in triplicate.
4.8 Characterization of the activation state of human hepatic stellate cells
Quiescent hepatic stellate cells accumulate 80% of retinol as retinyl palmitate in lipid 
droplets in the cytoplasm, and can release it depending on the extracellular retinol state 
(18). However, following liver injury, stellate cells can become activated, proliferate, lose 
retinol droplets, and secrete extracellular matrix (ECM), becoming myofibroblast-like cells. 
In this activated state, they may be responsible for the progression of liver disease to 
fibrosis (19).
In stellate cells isolated from patients we will evaluate the expression of genes involved in fibrogenic 
processes, such ascollagen-1, ³-smoothmuscleactin (-SMA),transforminggrowthfactor (TGF) ³1, 
tissueinhibitormatrixmetalloproteases (TIMP) andmatrixmetalloprotease (MMP) 13 in order to 
investigate its activation status and evaluate any differences determined by the variants of PNPLA3.
However, stellate cells do not release retinol or its metabolites only during fibrogenic 
processes, but also during regenerative processes, such as after partial hepatectomy (20). In 
fact, these cells possess enzymes for the storage and metabolism of retinol. Retinol 
acquired from outside is preferentially stored as retinyl esters of retinol by an enzyme
lecithin retinol acyl-transferase (LRAT) using acyl-CoA, or fromretinol acyltransferase (ARAT) 
in the case of quiescent cells (21). Retinol can then be metabolised into retinaldehyde by
alcohol dehydrogenase (ADH) and subsequently be converted to retinoic acid (RA) by
retinaldehyde dehydrogenase (RALDH) (22).
To better understand retinol metabolism in stellate cells isolated from patients with different 
PNPLA3 genotypes, we will investigate the expression of genes such as LRAT, ARAT, ADH and 
RALDH, involved in retinol storage and release.
Furthermore, depending on their activation state, stellate cells can regulate the progression of liver 
disease also by supporting inflammatory processes, producing cytokines, chemokines and/or

by directly interacting with T cells, natural killer (NK) and NKT cells (23). Therefore, we will also 
investigate the expression of these genes that contribute negatively or positively to the resolution of 
liver damage in assembloids.
5. ENDPOINTS
5.1 Primary Endpoints
1. Isolation of epithelial cells in order to generate models of the main populations of liver cells in a 
three-dimensional culture environment, called organoids, and molecular characterization of the 
generated models. Isolation of stellate cells and their phenotypic and molecular characterization. 
Generation of assemblyloids that mimic the hepatic tissue architecture and its phenotypic and 
molecular characterization to study the onset and development of steatohepatitis.
2. Evaluation of the impact of the I148M variant (evaluation of possible combinations both in 
epithelial cells - hepatocytes and in stellate cells) on the steatohepatitic phenotype in human liver 
assembloids, with particular reference to:
" Lipid accumulation in hepatocytes (quantitative and qualitative analysis)
" Lipotoxicity and expression of inflammatory markers (gene expression)
" Activation of fibrogenesis (assessment of the activation state of stellate cells, retinol 
metabolism and extracellular matrix deposition)
Steatohepatitis will be defined in the simultaneous presence of all these alterations in the 
assemblage.
3. Analysis of variants of the TM6SF2, MBOAT7 and GCKR genes, previously correlated with the 
development of non-alcoholic steatohepatitis (14).
Secondary Endpoints
- Knowledge of the behavioral and morphological differences between physiological and NASH 
hepatocytes and stellate cells.
- Detection of new biomarkers predictive of the development and progression of the 
disease.
a. - Analysis of epigenetic differences in physiological conditions, onset and progression of 
NASH.

- Knowledge of the molecular mechanisms underlying cellular-level observations using 
<omics= scale assessments and CRISPR-Cas9 genetic engineering.
- Analysis of the impact of PNPLA3 silencing on the steatohepatic phenotype of 
human liver assembloids, stratified by I148M variant carrier status in epithelial cells 
vs. stellate cells.
6. DURATION / TIMELINE OF THE STUDY
Study start month and year: 07/2021 
Enrollment close month and year: 07/2024 
Study end month and year: 07/2026
The study will last 60 months. In the first three years we expect to enroll approximately 60 subjects in 
accordance with the inclusion criteria.
PROJECT GANTT
Intervention
Period (months)
1-12
13-24
25-36
37-60
Enlistment
-
-
- -
Informed consent
-
-
-
Criteria of
inclusion/exclusion
-
-
-
Generation of organoids
-
-
-
Expression Evaluation
Genetics in organoids
hepatic
- -
- -
- -
Evaluation of specifications
liver proteins
in liver organoids
- -
- -
- -
Differentiation to
hepatocytes of the
-
- -
- -
-

organoids
Isolation and
characterization of
star cells
- -
- -
- -
-
Generation of
assemblyloids
-
- -
- -
- -
In vitro treatment with
fatty acids,
cholesterol and sugars, 
etc.
-
- -
- -
- -
Therapeutic silencing
PNPLA3 in
assemblyloids
-
-
-
- -
Correlation analysis
data
-
-
-
7.STATISTICAL ANALYSIS
7.1 Sample size and data analysis
The sample size was estimated in view of achieving the two primary outcomes of the
project.
Previously published works (24, 25, 26, 27) have shown that the generation efficiency of liver 
organoids is approximately 100% starting from surgical resections and 33% from biopsies (24,
25).
Considering these estimates, in order to isolate at least 20 independent organoid cultures of each 
type of condition of interest in our study, we estimate to recruit at least 30 donors for each genetic 
variant. The intent is to obtain replicates for the analyses with a sufficient representation of the 
different stages of liver damage (normal liver, inflammation/fibrosis and hepatocellular carcinoma) 
and to be able to map the impact of the main most common inherited genetic variants (present in at 
least 20% of individuals) in the population.
For the first primary outcome, the primary analyses will be conducted from single-individual cell 
cultures. Given the descriptive nature of mapping cellular phenotypes and gene expression, and 
the large number of organoids (>10^3)   and cells (>10^9) from a single individual, we will have 
sufficient statistical power (>80%) to identify and analyze specific cellular subsets representing 
>1% of the overall population.
As regards the second primary outcome, although there are important limitations in the estimate 
inherent to the type of project and linked to the uncertainty regarding the final characteristics of the 
model that will be developed during the project, we expect to observe a progressive increase in the

presence and severity of the steatohepatitis phenotype in organoids with increased number of PNPLA3 
I148M variants. In particular, we estimated that we could reach a statistical power of 95% (alpha 0.05, 
two-tailed test) to observe a frequency of steatohepatitis phenotype of 75% vs. 25% in assemblyloids with 
extreme PNPLA3 genotypes, homozygous for the at-risk variant vs. wild-type (20 cases vs. 20 controls), 
which is consistent with the increased risk observed in clinical cohorts (28).
Statistical analyses will be performed using JMP (SA, Cary, NC) and R (https://www.r-
project.org) software and statistical comparisons will be made using the one-way ANOVA or 
chi-square test, where appropriate. P values   < 0.05 will be considered statistically 
significant.
8. ADVERSE EVENTS
The project does not involve the administration of drugs or other substances or ad hoc invasive interventions 
outside of normal clinical practices. Therefore, no adverse events are expected.
9. RISK/BENEFIT ASSESSMENT
The study does not foresee an immediate benefit for patients, but the results of this trial will have 
the potential to lead to a decoding of the mechanisms underlying the development of NASH, resolve 
the temporal order of events that regulate its evolutionary trajectory, understand its progression, 
characterization and improve the therapeutic management of patients.
10. STUDIO MANAGEMENT
10.1Data collection and management
Each participant will be assigned a unique code at enrollment. The file that associates the 
participant code with the participant identification data will be stored separately on a password-
protected computer. The study database will be password-protected and uploaded to a 
password-protected computer and accessible only to study personnel designated by the 
principal investigator. De-identification of the data will be done in such a way that researchers 
accessing the database will not be able to trace the identity of the subjects in any way. Only local 
investigators will be able to trace the identity of the enrolled subjects.
10.2Regulatory aspects and ethical considerations

10.2.1 Approval by the Competent Authority
In accordance with applicable regulations, the principal investigator must obtain approval from the 
appropriate Competent Authority prior to initiating the clinical study.
This study will be conducted in accordance with the ICH/GCP (International Conference of
Harmonization/GoodClinicalPractice) and all applicable laws, including the Helsinki Declaration of 
June 1964, as amended by the last World Medical Association General Assembly in Seoul, 2008.
10.2.2 Ethics Committee Approval
The investigator must ensure that the protocol has been reviewed and approved by the local independent 
Ethics Committee (EC) before starting the study.
The CE must also review and approve the informed consent (IC) form and all written information 
received from the patient prior to enrollment in the study.
If it is necessary to modify the protocol and/or the IC during the study, the investigator will be the 
guarantor and therefore the person responsible for ensuring the review and approval of such modified 
document as requested by the CE.
The content of these changes will be implemented only after the CE has approved them. Until then, 
it will be necessary to refer to the previous version of the document already approved.
10.2.3 Informed consent (IC)
The investigator or other personnel designated by him have the task of informing people about all aspects and 
procedures of the study.
The process for obtaining informed consent must comply with applicable regulatory 
procedures. The investigator (or designated staff member) and the subject must date and sign 
the informed consent form prior to the patient initiating any study-related procedures. The 
subject will receive a copy of the IC dated and signed by both parties; the original copy will be 
retained in designated study archives. Neither the investigator nor designated staff member 
should in any way coerce or influence a subject to participate or continue to participate in the 
study. A subject's decision to participate in the study must be completely voluntary. The 
investigator and designated staff member should emphasize to the subject that he or she may 
withdraw consent at any time without penalty or loss of any benefits to which the subject may 
be entitled.
The isolated biological material may be used for research purposes only following the informed 
consent expressed by the recipient of the transplanted organ..Written or oral information relating to 
the study, including the written consent form, must not contain any linguistic expressions that

forces the subject to waive (even apparently) his or her legal rights, or that would exonerate the 
investigator, agency, or sponsor from liability for negligence.
10.3Duties of the experimenter
In accordance with applicable local regulations, the investigator must submit periodic 
reports regarding the progress of the study at his/her site to the CE and notify the CE of the 
study closure. Periodic reports and closure notification are part of the investigator's 
responsibilities.
10.4 Study monitoring
In accordance with applicable regulations and good clinical practice (GCP), the monitor must visit or 
contact the center periodically. The duration, nature and frequency of such visits / contacts depend 
on the recruitment frequency, the quality of the documents held by the center and their adherence 
to the protocol.
Through these contacts, the monitor must:
" monitor and evaluate the progress of the study
" examine the collected data
" conduct source document verification
" identify each problem and related solutions
The purposes of the monitoring activity are to verify that:
" the rights and well-being of the subject are respected
" the study data are accurate, complete and verifiable from the original documents
" the study is conducted in accordance with the protocol and any approved amendments, GCP and 
applicable regulations
The experimenter must:
" give the monitor direct access to all relevant documentation
" dedicate part of his time and his staff to the monitor to discuss the monitoring results and any 
other possible aspects.

The monitor must also contact the center prior to the start of the study to discuss the protocol and data 
collection procedures with the staff.
10.5Study quality assurance
As the Sponsor, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico may, at its 
discretion, carry out a quality control of the study. In this case, the investigator must allow 
the monitor direct access to all relevant documentation and dedicate part of his/her time 
and personnel to the reviewer to discuss the results of the monitoring and any other 
aspects of the study.
In addition, Regulatory Authorities may conduct inspections. In this case, the investigator must 
allow the inspector direct access to all relevant documentation, and dedicate part of his time 
and personnel to the inspector to discuss the monitoring results and any other aspects of the 
study.
10.6 Closing of the study
At the time of study closure, the monitor and the experimenter must activate a series of 
procedures:
" review all study documentation
" reconcile study data
" reconcile all clarifying reports.
10.7 Document storage
In accordance with current national regulations, the investigator must keep a copy of all 
documentation and store it in a dry and safe place after the study has been closed.
10.8 Disclosure of information regarding scientific discovery
10.8.1 Confidentiality
The investigator and other personnel involved in the study must handle all information relating to the 
study (including the protocol, data obtained and all documentation generated during the study) and must 
not use such information, data or reports for purposes other than those described in the

protocol.
These restrictions do not apply to:
1) information that becomes publicly available, not due to negligence on the part of the 
investigator or his staff;
2) information that requires confidential disclosure to CE for the sole purpose of evaluating the study;
3) information that must be disclosed in order to obtain appropriate medical care for a 
study subject.
10.8.2 Publications
The Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico is the sole owner of the data.
10.9 Intellectual property rights on the results of the study
The Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico is the sole owner of the intellectual 
property rights on the study.
11. INDEMNITY AND COMPENSATION IN CASE OF DAMAGES
In case of unwanted events or any damages that may arise from participation in research, 
our Institute's Insurance Policy also extends to cover subjects participating in research 
projects.
12. FINANCIAL AGREEMENTS
The costs of the study procedures exceeding normal clinical practice will be entirely covered by 
funds deriving from the Horizon2020 3 Europa EU funding, Grant H2020-ICT-2020-2 Project Number 
101016726
The costs of immunohistochemical analyses of liver histology samples to be carried out at the Institute for 
Liver and Digestive Health, University College London, will be covered in full by the Institute for Liver and 
Digestive Health

13. DISCLOSURE ON CONFLICTS OF INTEREST
The experimenters declare no conflicts of interest.
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