Development of an In Vitro Human Liver System for Interrogating Non-Alcoholic. Brian R. Wamhoff, 501 Locust Ave., Charlottesville, VA 22902

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1 Supplemental Information Development of an In Vitro Human Liver System for Interrogating Non-Alcoholic Steatohepatitis Ryan Feaver, Banumathi K Cole, Mark J Lawson, Stephen A Hoang, Svetlana Marukian, Robert A Figler, Arun J Sanyal, Brian R Wamhoff, Ajit Dash HemoShear Therapeutics LLC, Charlottesville, VA Virginia Commonwealth University School of Medicine Richmond, VA Corresponding Author: Brian R Wamhoff, 5 Locust Ave, Charlottesville, VA 9 Wamhoff@HemoShearcom

2 Supplemental Figures and Tables: Supplemental Figure Correlation plot demonstrating the reproducibility of data from different experiments in the lipotoxic system Hepatocyte transcriptomic data of all genes (each gene is an individual dot) from two different experiments (# and #) performed months apart with n=3 and n=4 donors, respectively, is represented in this correlation plot Comparison is healthy vs lipotoxic + ng/ml TNFα milieu for each experiment The scatterplot shows that genes from both experiments exhibit a strong positive correlation in gene response, and thus high reproducibility (Log Fold Change) Experiment # (Log Fold Change) Experiment # LGLI NASH BGA3 Pearson correlation = at a p value <e-6

3 Fatty Acid Biosynthesis Oxaloacetate PC TCA Cycle Fatty Acid Pyruvate ACLY Citrate ACSL3 ACSL ACAS ACACB ACACA Acetyl-CoA MLYCD ACSL4 ACSL5 ACSL6 Malonyl-CoA CPTA FASN Palmitic Acid Acyl-CoA Cytosol Cytosol ELOVL6 Steric Acid SCD Oleic Acid Acetyl-CoA CPTA Long-Chain fatty acid Beta Oxidation ACSL ACSL3 ACSL4 ACSL5 Mitochondrion logfold ACSL6 ACAS Fatty acyl CoA DGAT DGAT Triacylglyceride Synthesis pathway Supplemental Figure Transcriptomic data from hepatocytes exposed to the lipotoxic compared to the healthy milieu is overlaid onto a diagram representing the fatty acid biosynthesis pathway A red-filled box indicates the gene is upregulated while a blue-filled box indicates the gene is downregulated; the intensity of the color corresponds to greater logfoldchanges Boxes with a green perimeter indicate the gene expression logfoldchange is significant (FDR<%) 3

4 Cholesterol Biosynthesis Fatty Acid Degradation Acetyl-CoA HMGCS HMG-CoA Cholesterol HMGCR DHCR7 Mevalonic acid 7-Dehydrocholesterol MVK SC5DL Mevalonic acid-5p Lathosterol PMVK Mevalonic acid 5-pyrophosphate NSDHL SC4MOL CYP5A Dimethylallylpyrophosphate IDI MVD isopentenyl pyrophosphate Lanosterin LSS logfold FDPS (S)-,3-Epoxysqualene Geranyl-PP farnesyl pyrophosphate Squalene SQLE FDPS FDFT Supplemental Figure 3 Transcriptomic data from hepatocytes exposed to the lipotoxic compared to the healthy milieu is overlaid onto a diagram representing the cholesterol biosynthesis pathway A red-filled box indicates the gene is upregulated while a blue-filled box indicates the gene is downregulated; the intensity of the color corresponds to greater logfoldchanges Boxes with a green perimeter indicate the gene expression logfoldchange is significant (FDR<%) 4

5 Glycolysis/Gluconeogenesis Glucose SLCA SLCA SLCA3 SLCA4 Glycolysis Gluconeogenesis SLCA5 Glucose HK HK HK3 G6PC GCK Glucose-6P Pentose Phosphate Pathway GPI Glycogen metabolism PFKM PFKL PFKP Fructose 6P Fructose-,6BP FBP FBP ALDOA ALDOB ALDOC Glyceraldehyde 3P Dihydroxyacetone-P Triglyceride synthesis GAPDHS GAPDH TPI,3BP-Glycerate PGK PGK Cytosol 3P-Glycerate PGAM Mitochondrion PGAM P-Glycerate ENO Aspartate Aspartate ENO3 P-enolpyruvate ENO GOT GOT PKM PKM Oxaloacetate Oxaloacetate PKLR PCK MDH PC MDH LDHAL6B Pyruvate Malate Malate logfold LDHA LDHB LDHC Lactate MPC MPC Pyruvate PDHA PDHA PDHB TCA Cycle DLAT DLD PDHX Acetyl-CoA Supplemental Figure 4 Transcriptomic data from hepatocytes exposed to the lipotoxic compared to the healthy milieu is overlaid onto a diagram representing the glycolysis/gluconeogenesis pathway A red-filled box indicates the gene is upregulated while a blue-filled box indicates the gene is downregulated; the intensity of the color corresponds to greater logfoldchanges Boxes with a green perimeter indicate the gene expression logfoldchange is significant (FDR<%) 5

6 Count 4 Value Gene Response Heatmap Gene Response Heatmap Gene Gene Response Heatmap Heatmap Gene Response Heatmap Gene Response Heatmap Gene Response Heatmap Count 6 Value Count 4 Value Count Count 6 Value Value Count Value FADD NUMA PTK BID CASP7 SPTAN RIPK TNFRSFB FAS Apoptosis cell CYPA CAT HADHA TXNRD NOX4 HADHB MGST XDH ACADS NQO CASP6 NFKBEC HEALTH ACADM SATB GPX TNFRSFA MAPK4 ECHS CASP MADD NFIX TRADD SOD3 ECI CASP8 UGTA6 APAF CASP MAPK DECR DIABLO GSR CRADD GCLC Oxidative Stress Response DFFA SOD TNFRSFA TOP SP CASP9 TXNRD CASP3 CYBA MAP3K TXN CYCS GAS NFEL DFFB SOD BAX JUNB KRT8 BCLL FOS VIM GPX3 TNFSF HMOX APP MAOA GSN LIMK MTX Mitochondrial Fatty Acid Beta Oxidation Genes (97) comparison MUT ACADL PCCA MCEE PCCB HADH Genes (9) ACADVL Inflammation Genes (9) comparison cell CXCL6 CCR6 CCL6 CXCL TLR4 CXCL CXCL8 TLR CCL TLR STAT ILRB CXCL6 IFNAR CCL5 STAT6 IFNGR STAT5A CSF STAT ILR STAT5B MIF IL5 IFNAR TLR3 TNFRSFB IFNLR IL7 TNFSF3B ILRN CXCR5 IFNGR TLR6 TLR5 STAT3 AIMP CXCL CXCL3 Genes (86) Inflammasome Genes (86) PTGS EDN PTGS comparison cell cell PYCARD PRX7 TXNIP CASP BCLL NFKB APP NFKB TXN RELA PANX HSP9AB SUGT Genes (73) logfold Genes (73) cell EC SMC cell_type i p Responses (NA) Responses (NA) Responses (NA) (NA) Responses (NA) Responses (NA) Responses (NA) HSP9AB CALM HSP9B CAV Supplemental Figure 5 Transcriptomic data from hepatocytes NOS3exposed to the lipotoxic compared to the healthy milieu is presented KLFfor various biological pathways associated with cellular stress and inflammation KLF4 Expression of each gene is represented as logfold-change of lipotoxic vs ARG healthy CCA STATIC milieu CCA STATIC (red=upregulation, ICS CCA ICS CCA CCA STATIC blue=downregulation) CCA STATIC ICS CCA ICS CCA n=6 experiments, 3 donors 6

7 IL8 (pg/ml) MCP(pg/ml) CULTURE: MILIEU: < INTERACTION: < Y Y H H + TNF CULTURE: < MILIEU: < INTERACTION: 8 IL6 (fg/ml) p= p=3 YKL4 (pg/ml) CULTURE: < MILIEU: < INTERACTION: TNF CULTURE: < MILIEU: < INTERACTION: 76 CXCL (pg/ml) VEGF (pg/ml) CULTURE: 53 MILIEU: < INTERACTION: TNF CULTURE: 4 MILIEU: 8 INTERACTION: 4 Mono-culture Multi-culture 8 64 Y Y H H + TNF + TNF + TNF Supplemental Figure 6 The presence of non-parenchymal cells (NPCs) in the in vitro human liver system significantly impacts its inflammatory profile Secreted analytes were measured in the media effluent from devices at day The lipotoxic milieu for these experiments is composed of 65µM oleic acid and ng/ml TNFα n=4 experiments, donor Triangles indicate samples were below lower limit of quantification Tables above charts provide overall significance from -way ANOVA for cell culture and milieu treatment conditions and the interaction p-value Multiple comparison were performed as indicated with p<5, p<, -way ANOVA, Tukey 7

8 Mono-culture Milieu Mono-culture Milieu 5 Mono-culture PC PC Multi-culture Milieu Milieu 5 Multi-culture Milieu Multi-culture Milieu 5 5 PC PC Supplemental Figure 7 The presence of non-parenchymal cells (NPCs) in the in vitro human liver system significantly impacts the transcriptomic profile of hepatocytes Transcriptomic data was acquired from hepatocytes exposed for days to a lipotoxic milieu composed of 65µM oleic acid and ng/ml TNFα in the presence or absence of NPCs n=3-6 experiments, donor Gene expression profile reveals that the hepatocytes from healthy and lipotoxic treatments separate along principle component, and these further separate along principle component if co-cultured with NPCs 8

9 CE CER DAG DCER FFA HCER LCER LPC (OCA) (VEH) logfold LPE PC PE PI RSI SM TAG 4 4 logfold (VEH) (VEH) Supplemental Figure 8 OCA treatment in the lipotoxic milieu promotes a change in lipid profile towards the healthy controls Lipids from hepatocytes from device exposed to the healthy or lipotoxic milieu with 5µM OCA or vehicle control were measured by metabolomics Scatterplot representation of differentially expressed lipids in these hepatocytes are shown as logfold-change and colored by RSI n=4 experiments, donors Each lipid species is represented in an individual plot: CE (cholesterol ester), CER (ceramide), DAG (diacylglycerol), DCER (dihydroceramide), FFA (free fatty acid), HCER (hexosylceramide), LCER (lactosylceramide), LPC (lysophosphatidylcholine), LPE (lysophosphatidylethanolamine), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PI (phosphatidylinositol), SM (sphingomyelin), TAG (triacylglycerol) 9

10 p= Total Intracellular Cholesterol (Fold VEH) 8 Veh OCA ATOR Supplemental Figure 9 Obeticholic acid (OCA) increases total intracellular cholesterol levels Total intracellular cholesterol was measured from hepatocytes from the device exposed to vehicle control, 5µM OCA, or 3nM atorvastatin (ator) in the lipotoxic milieu for days and represented as fold change relative to vehicle controls Atorvastatin serves as a positive control as it inhibits HMG-CoA reductase, the rate-limiting enzyme of the cholesterolproducing mevalonate pathway n=4 experiments, 4 donors p<5, student s -tailed t-test

11 In Vitro References / Figure/Table Model Reviews Steatosis / Lipogenesis Yes Fig -3 Hypertriglycermia Yes Table -5 Increased Cholesterol Yes Table -5 Increased Glucose Output Yes Fig 3A,3 6 Insulin Resistance Yes Fig 3-3,6,7 Cell Stress / Elevated ALT Yes Fig 4-3,7,8 Inflammatory Signaling Yes Fig 4,3,7,8 Increased Apoptosis Yes Fig 4,3,7-9 Histological Features (eg ballooning) NA -3 Early Fibrosis Yes Fig 5,3,7, Advanced Fibrosis Fig 5,3, Cirrhosis NA,3, Oncogenesis NA,3 Supplemental Table Pathological features of NAFLD in the in vitro human liver lipotoxic system

12 Donor Donor ID Age Gender BMI Vendor HC5-3 Male 5 XenoTech QHuf44 55 Female 9 QPS 3 QHuf58 58 Female QPS 4 HC3-3 4 Female 3 XenoTech 5 QHu Male 3 QPS Supplemental Table Hepatic donors used in this study with their sourcing details and demographic information

13 Supplemental References: () Sass DA, Chang P, Chopra KB Nonalcoholic fatty liver disease: a clinical review Dig Dis Sci 5;5:7-8 () Paschos P, Paletas K Non alcoholic fatty liver disease and metabolic syndrome Hippokratia 9;3:9-9 (3) Marra F, Lotersztajn S Pathophysiology of NASH: perspectives for a targeted treatment Curr Pharm Des 3;9: (4) Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt, MD, Parks EJ Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease J Clin Invest 5;5: (5) Puri P, Baillie RA, Wiest MM, Mirshahi F, Choudhury J, Cheung O, et al A lipidomic analysis of nonalcoholic fatty liver disease Hepatology 7;46:8-9 (6) Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui, JM, Fung C, et al NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome Hepatology ;35: (7) Basaranoglu M, Basaranoglu G, Senturk H From fatty liver to fibrosis: a tale of "second hit" World J Gastroenterol 3;9:58-65 (8) Takaki A, Kawai D, Yamamoto K Multiple hits, including oxidative stress, as pathogenesis and treatment target in non-alcoholic steatohepatitis (NASH) Int J Mol Sci 3;4:

14 (9) Wieckowska A, Zein NN, Yerian LM, Lopez AR, McCullough AJ, Feldstein AE In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease Hepatology 6;44:7-33 () Friedman SL Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver Physiol Rev 8;88;5-7 4