Fig 2: Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables identification of all hepatic cell types including bona fide cell doublets, related to Figure 1(A and B) Top DEGs (A) and DEPs (B) for cell types from Figure 1B.(C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocols; 71,162 cells from ex vivo digestions, 96,066 cells from in vivo digestions, and 18,666 nuclei. Numbers on plots represent numbers of cells/nuclei per population.(D) Correlation plots showing genes captured within the KC, B cell and neutrophil populations with and without addition of CITE-seq antibodies.(E) Expression of VSIG4, CD206, and ESAM (protein, top) and Vsig4, Mrc1, and Esam (mRNA, bottom).(F) UMAP showing clusters of cells when only minimal QC for gene number and % mitochondrial genes is performed; 17,669 cells pooled from 3 samples. Expression of Cd5l, Cd19, and Kdr by the clusters facilitating identification of cell types per annotation.(G) CITE-seq data from (F) in Flow-Jo showing expression of CD206 and ESAM in total KCs (left) and total B cells (middle). Numbers represent % of entire KC or B cell population. Identified populations were then mapped back onto the original UMAP (right).(H) Expression of CD31, CD26, and CD38 by indicated populations.(I) Heatmaps showing expression of top DEGs between KC1s and LSECs (left), KC2s and KC1s + LSECs (middle) and B cell2s and B cell1s + LSECs (right).(J) 3D reconstruction of murine liver following perfusion with antigen fix to inflate endothelial cells and staining with antibodies against CD31, CD206, and F4/80.(K) UMAP showing clusters generated from Visium analysis of liver tissue (4 samples) and liver capsule (1 sample).(L) Top unbiased genes defining zonation trajectory from portal to central vein in Visium.(M) Expression of Glul and Epcam by confocal microscopy (left), annotation of portal, periportal, mid, and central regions on same tissue section (middle) and overlay of both datasets (right).(N) Identification of cholangiocyte (left) and cDC (right) signatures on zonated Visium spots.(P) Molecular Cartography showing expression of indicated zonated hepatocyte mRNAs in liver tissue. Data are representative of 2 mice.(O) Expression of Itgae (encoding CD103) in the UMAP of the total liver (left) and flow cytometric analysis of total cDC1s for CD103 and MHCII expression in the healthy murine liver (right).

Fig 3: Identification of bona fide Kupffer cells across species(A) Cells/nuclei were isolated from liver biopsies (∼1–2 mm3; 14 cells, 5 nuclei) from patients undergoing either liver resection, cholecystectomy or gastric bypass. Live cells/intact nuclei were FACS-purified. Either total live, live CD45+, and live CD45− or live CD45+, CD3−, and CD19− cells were sorted. 7 cell samples were stained with a panel of 198 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC, 167,598 cells/nuclei were analyzed using TotalVI.(B) UMAP of sc/snRNA-seq data.(C) UMAP of Visium data from 4 patient biopsy samples.(D) Split of Visium spots based on % steatosis.(E) Healthy and steatotic Visium liver tissue with clusters overlaid and H+E staining to identify steatotic zones.(F) Zonation of Visium data (top) with zonation pattern mapped onto liver tissue (bottom).(G) Indicated cell signatures from sc/snRNA-seq mapped onto Visium zonation trajectory, healthy (top), steatotic (bottom).(H) Myeloid cells (40,821 cells) were isolated from Figure 4B and re-clustered with TotalVI.(I) Expression of VSIG4 protein (top) and CD5L mRNA (bottom).(J) Expression of VSIG4, F4/80, FOLRB, and GLUL combined with Cd5l/CD5L on murine (left) and human (H25; right) livers. Scale bars, 50 μm. Inset in bottom panels. Scale bars, 20 μm. Images are representative of 2–4 livers.(K) Livers (2/species) were isolated from healthy macaque, pig, chicken, hamster, and zebrafish. Cells were isolated by ex vivo digestion for CITE-seq (pig; 198 human antibodies) or scRNA-seq (hamster, chicken, and zebrafish), or nuclei were isolated for snRNA-seq (macaque). Total live cells (hamster, chicken, and pig), DsRed+GFP+ cells (zebrafish) or nuclei (macaque) were FACS-purified. Following QC, 8,483 nuclei (macaque) or 21,907 (pig), 5,965 (hamster), 7,457 (chicken), and 4,957 (zebrafish) cells were analyzed using TotalVI (pig) or scVI (macaque, hamster, chicken, and zebrafish) (top). KCs were identified using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019) (bottom). See also Figures S6 and S7 and Tables S1, S2,S3, S5, S6,S8, and S9.

Fig 4: A proteogenomic atlas of the healthy murine liver(A) Hepatic cells were isolated from healthy C57B/l6 mice by ex vivo (5 mice, 15 samples) or in vivo (5 mice, 19 samples) enzymatic digestion. Alternatively, nuclei were isolated by tissue homogenization (4 mice, 12 samples). Live cells/intact nuclei were FACS-purified. For cells, total live, live CD45+, live CD45−, live hepatocytes, or myeloid cells (live CD45+, CD3−, CD19−, B220−, NK1.1−) were sorted. 18 samples (7 ex vivo, 11 in vivo) were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC 185,894 cells/nuclei were clustered using TotalVI.(B) UMAP of sc/snRNA-seq data.(C) Tissue and capsule images from Visium analysis with clusters overlaid.(D) UMAP of zonation of Visium spots (left) and origin of the cells (right).(E) Zonation pattern mapped onto tissue slice.(F and G) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data.(H) mRNA zonation pattern in Visium highly multiplexed protein analysis and VSIG4-ADT expression pattern (left) and zonated expression patterns of indicated antibodies (right).(I) MICS analysis of indicated proteins.(J) Molecular Cartography of indicated genes and cell types.(K) mRNA (Xcr1, Flt3l, Mafb, and Clec10a) and protein (MHCII and F4/80) expression in the same tissue slice. Scale bars, 50 μm. PV, portal vein; CV, central vein. Arrows indicate specific cell types, colors correspond to cell type/markers. Images are representative of 2–4 mice. See also Figure S2 and Tables S1, S2, S3, and S5.

Fig 5: Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas, related to Figure 4(A) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 27,398 cells) were isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) for the cell types from Figure S5A.(D) CITE-seq data from Figure S5A were exported as an FCS file and an in silico gating strategy identified in FlowJo.(E) Human lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 105,790 cells) were isolated from Figure 4B and re-clustered with TotalVI.(F and G) Top DEGs (F) and DEPs (G) for the cell types from Figure S5E.(H) CITE-seq data from Figure S5E were exported as an FCS file and an in silico gating strategy identified in FlowJo.(I) Proportion of indicated cell types arising from patients with <10% (purple) or >10% steatosis (yellow).(J) Hepatic cells were isolated from 22 C57B/l6 mice fed either a standard diet (SD) or a western diet (WD) for 24 or 36 weeks to induce NAFLD and NASH by ex vivo (10 samples) or in vivo (12 samples) enzymatic digestion. Alternatively, livers were snap frozen and nuclei isolated by tissue homogenization (14 samples). Live cells/intact nuclei were purified using FACS. For cells, total live, live CD45+, live CD45−, live hepatocytes or myeloid cells (live CD45+, CD3−, CD19−, B220−, NK1.1−) were sorted. 10 samples were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC 121,980 cells/nuclei were clustered using TotalVI.(K) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 21,322 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI.(L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow).(M) Top DEGs between CTLs isolated from mice fed the SD (purple) or WD (yellow).