Direct contact between iPSC-derived macrophages and hepatocytes drives reciprocal acquisition of Kupffer cell identity and hepatocyte maturation
- Innovations in Food and Chemical Safety Programme (IFCS), A*STAR, Singapore
- Singapore Immunology Network, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore
- Institute of Bioengineering and Bioimaging, Singapore
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore
- NUS Graduate School – Integrative Sciences and Engineering Programme (ISEP), Singapore
- Mechanobiology Institute, Singapore
- CAMP IRG, Singapore-MIT Alliance for Research and Technology, Singapore
- Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine, China
- Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, Singapore
- U1356 Next Generation Immuno-Oncology research, Université Paris-Saclay, Gustave Roussy, France
Figures
Figure 1 with 2 supplements
Model development and effects of iMacs on iHeps.
(A) Schematic of experimental layout. (B) Principal component analysis of bulk RNAseq data from Day 0 iHeps, Day 3 co-iHeps, Day 7 co-iHeps, and Day 7 iHeps. (C) Volcano plot showing differentially-expressed genes 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. Error bars represented S.D., n=5 independent experiments. Student’s t-test was applied. *p<0.05, **p<0.01.
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Figure 1—source data 1
Differentially expressed genes between the iHeps samples.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig1-data1-v1.xlsx
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Figure 1—source data 2
Ingenuity pathway analysis for genes upregulated in Day 7 iHeps vs Day 7 co-iHeps.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig1-data2-v1.xlsx
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Figure 1—source data 3
Ingenuity pathway analysis for genes upregulated in Day 7 co-iHeps vs Day 7 iHeps.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig1-data3-v1.xls
Figure 1—figure supplement 1
Optimisation of co-culture conditions and validation of purity.
(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) Flow cytometry showing purity of macrophages by CD45, CD14, CD11b, CX3CR1, and CD163 before co-culture. (F) Flow cytometry showing relative cellular size and granularity of iMac and iHep populations after co-culture. (G) Expression of Albumin in Day 7 co-cultured iHeps vs iMacs. (H) Expression of CSF1R in Day 7 co-cultured iMacs vs iHeps.
Figure 1—figure supplement 2
Correlation of iPSC-derived hepatocytes with or without co-culture with iPSC-derived macrophages.
(A) Pearson correlation of iHeps against fetal hepatocytes from Popescu et al., 2019. (B) Top 40 genes influencing Pearson correlation of panel A. (C) Expression of ATF4, CASP3, and CASP9 showed no significant upregulation of stress or apoptosis genes after co-culture.
Figure 2 with 1 supplement
Effects of iHeps on iMacs.
(A) Principal component analysis of bulk RNAseq 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 and uniquely 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 differentially-expressed genes 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. Error bars represented S.D., n=5 independent experiments. Student’s t-test was applied. *p<0.05, **p<0.01, ***p<0.001.
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Figure 2—source data 1
Differentially expressed genes between the iMac samples.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data1-v1.xls
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Figure 2—source data 2
Ingenuity pathway analysis for genes upregulated in Day 7 co-iMacs vs Day 0 iMacs.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data2-v1.xls
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Figure 2—source data 3
Ingenuity pathway analysis for genes upregulated in Day 0 iMacs vs Day 7 co-iMacs.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data3-v1.xls
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Figure 2—source data 4
Ingenuity pathway analysis for genes upregulated in Day 7 cond-iMacs vs Day 7 co-iMacs.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data4-v1.xls
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Figure 2—source data 5
Ingenuity pathway analysis for genes upregulated in Day 7 co-iMacs vs Day 7 cond-iMacs.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data5-v1.xls
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Figure 2—source data 6
GO enrichment results for DEGs between Day 7 conditioned media iMacs and embryonic liver macrophages.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data6-v1.xlsx
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Figure 2—source data 7
GO enrichment results for DEGs between Day 7 co-cultured iMacs and embryonic liver macrophages.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig2-data7-v1.xlsx
Figure 2—figure supplement 1
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.
Figure 3
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. Error bars represented S.D., n=5 independent experiments. *p<0.05. Student’s t-test was applied. *p<0.05.
Figure 4
Changes in IL-6 level upon treatment with seven 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 independent experiments. One-way ANOVA was applied. *p<0.05, **p<0.01 between treatment and vehicle control.
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Figure 4—source data 1
Details of drugs used for testing.
- https://cdn.elifesciences.org/articles/108938/elife-108938-fig4-data1-v1.xlsx
Figure 5
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 independent experiments. One-way ANOVA was applied. *p<0.05, **p<0.01 between treatment and vehicle control.
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Direct contact between iPSC-derived macrophages and hepatocytes drives reciprocal acquisition of Kupffer cell identity and hepatocyte maturation
eLife 14:RP108938.
https://doi.org/10.7554/eLife.108938.3
