Model development and effects of iMacs on iHeps.

A) Schematic of experimental layout. B) Principal Component Analysis of bulk RNA-seq data from Day 0 iHeps, Day 3 co-iHeps, Day 7 co-iHeps and Day 7 iHeps. C) Volcano plot showing differentially-expressed genes (DEGs) between Day 7 iHeps and Day 7 co-iHeps. D) Differentially-expressed upregulated pathways between Day 7 iHeps and Day 7 co-iHeps. E) Gene expression levels of AFP, CYP2C9, IGF-2, EGR-1, ALDOB and HNF4A in Day 0 iHeps, Day 3 co-iHeps, Day 7 co-iHeps and Day 7 iHeps. Student’s T test was applied. *p<0.05, **p<0.01

Effects of iHeps on iMacs.

A) Principal Component Analysis of bulk RNA-seq data from Day 0 iMacs, Day 3 co-iMacs, Day 7 co-iMacs, Day 7 iMacs and Day 7 cond-iMacs. B) Heatmap showing the top 5 differentially (DEGs) and uniquely (UEGs) expressed genes from culturing iMacs alone, with conditioned media (cond-iMacs) and after co-culture with iHepatocytes (Day 7 co-iMacs). C) Volcano Plot showing differentially-expressed genes between Day 0 iMacs and Day 7 co-iMacs. D) Differentially expressed upregulated pathways between Day 0 iMacs and Day 7 co-iMacs. E) Volcano Plot showing DEGs between Day 7 cond-iMacs and Day 7 co-iMacs. F) Differentially expressed upregulated pathways between Day 7 cond-iMacs and Day 7 co-iMacs. G) Gene expression levels of LYVE1, ID1, ID3, RXRA, HBEGF and OSM in Day 0 iMacs, Day 3 co-iMacs, Day 7 co-iMacs, Day 7 iMacs and Day 7 cond-iMacs. Student’s T test was applied. *p<0.05, **p<0.01, ***p<0.001

Gene expression of iMac and iHep markers in co-cultures.

A) Expression of macrophage markers (left panel) and KC-specific markers (right panel) on day 3 and day 7 of co-culture. B) Expression of macrophage markers (left panel) and KC-specific markers (right panel) on day 7 of co-culture compared to iMac cultured in PHCM. C) Expression of hepatic markers (left panel) on day 3 and day 7 of co-culture. * p<0.05.

Changes in IL-6 level upon treatment with 7 paradigm compounds when iMac-derived KCs were used.

Cytokine production was assessed in iMac-derived iKC and iHep co-culture treated with the drug stated in the plot title. Data is expressed as the percentage of the LPS-treated vehicle control. Error bars represented S.D., n=3. One-way ANOVA was applied. *p<0.05, **p<0.01 between treatment and vehicle control.

Changes in IL-6 level upon treatment with 7 paradigm compounds without iMac-derived KCs.

Cytokine production was assessed in blood monocyte-derived macrophages and iHep co-culture treated with the drug stated in the plot title. Data is expressed as the percentage of the LPS-treated vehicle control. Error bars represented S.D., n=3. One-way ANOVA was applied. *p<0.05, **p<0.01 between treatment and vehicle control.

Optimisation of co-culture conditions and correlation of iPSC-derived hepatocytes with or without co-culture with iPSC-derived macrophages.

A & B) Fold change in expression of key hepatocyte genes in normal media volume and supplement concentration (A) or double media volume and supplement concentration (B), normalised to normal hepatocyte culture condition. C) Production of Albumin and Urea in co-culture system after 2 days, normalised to non-co-cultured hepatocytes. D) Flow cytometry of co-culture 3 and 7 days after co-culture. E) Pearson correlation of iHeps against fetal hepatocytes from Popescu et al, (2019).

Correlation of iMacs with embryonic liver monocytes and macrophages, and pathway analysis of the DEGs.

A) Pearson correlation of iMacs under control, conditioned media or co-culture conditions against embryonic liver monocytes and macrophages from Bian et al., (2020). B) Pathway analysis of DEGs between Day 7 conditioned media iMacs and embryonic liver macrophages. C) Pathway analysis of DEGs between Day 7 co-cultured iMacs and embryonic liver macrophages