Fig 2: ALK1-BMP9/10 axis regulates KC development(A) Mouse BM monocytes were cultured in the presence of CSF1 and indicated concentrations of human ac-LDL, prior to analyzed for expression of indicated genes by qPCR. Data are pooled from 2 experiments. One-way ANOVA with Bonferroni post-test compared with 0 ng/mL.(B) NicheNet circos plot highlighting conserved ligand-receptor pairs and induced target genes between KCs and indicated niche cells in human and mouse.(C) Feature plots showing expression of ALK1 (Acvrl1) in human myeloid cells (left) and GDF2/BMP10 in CD45- cells (right).(D) Livers were harvested from Fcgr1-CrexAcvrl1fl/fl mice or Acvrl1fl/fl or Acvrl1+/+ controls and KCs examined (left) and quantified (right) using VSIG4 expression.(E) Expression of indicated KC markers by mac populations in Fcgr1-CrexAcvrl1fl/fl or Acvrlfl/fl control mice. Data are pooled from 3 independent experiments with n = 9 per group. Student’s t test.(F) Expression of indicated markers in livers of Fcgr1-CrexAcvrl1fl/fl or Acvrl1fl/fl or Acvrl1+/+ control mice by confocal microscopy. Scale bars, 50 µm. Images are representative of 2 mice per group.(G) Schematic of chimera experiment setup.(H) % chimerism normalized to levels in blood Ly6Chi monocytes in Clec4f-Dtr mice 7 or 13 days after DT administration following partial irradiation and receiving either Acvrlfl/fl or Fcgr1-CrexAcvrlfl/fl BM 4 weeks earlier. Data are pooled from 2 independent experiments with n = 10–12 mice per group.(I) Schematic of Fc trap experiment setup.(J) Representative FACS plots showing VSIG4 and CLEC2 expression by total macs. Numbers represent % of total mac population in the indicated gate (left) and % of VSIG4+ and VSIG4- macs among total CD45+ cells in the different treatment conditions. One-way ANOVA with Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S8.

Fig 3: 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).

Fig 4: Conserved and unique features of KCs across species, related to Figure 4(A and B) Expression of human-murine KC signature genes across cell types in mouse (A) and human (B).(C) Unbiased identification of KCs in mouse and human using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019).(D–H) Annotated UMAPs from indicated species and expression of top KC-specific genes compared with other cells per species.(I) Expression of previously identified core murine transcription factors (Bonnardel et al., 2019) by KCs across species.(J) Venn diagram showing convergence and divergence of expression of top 50 KC genes per species across species, see Table S9 for genes lists per species.(K) Top DEPs (identified with cross reactive human antibodies) in the pig CITE-seq data.(L) Expression of VSIG4 in the porcine liver by confocal microscopy.(M) Expression of VSIG4, CD68 (protein), and CD5L (mRNA) in macaque liver. PV, portal vein; HA, hepatic artery; BD, bile duct. All images are representative of 2 livers.(N and O) Conserved expression of indicated genes across CD45- (N) and CD45+ (O) cell types and species.

Fig 5: Cell types identified in transcriptomic studies depend upon cell/nuclei isolation technique used, related to Figure 1Cells were isolated from livers of healthy C57B/l6 mice by either ex vivo or in vivo enzymatic digestion. Alternatively, livers were snap frozen and nuclei subsequently isolated following tissue homogenization by a sucrose gradient (3 mice per isolation method). Live cells/intact nuclei were identified and purified using flow cytometry. For the cells, either live CD45+, live CD45- or live hepatocytes were sorted. 1 ex vivo digested sample and 1 in vivo digested sample were also stained with a panel of 107 (ex vivo cells) or 161 (in vivo cells) oligo-conjugated antibodies for CITE-seq analysis. FACS-purified cells/nuclei were loaded onto the 10× chromium platform and scRNA-seq, CITE-seq, or snRNA-seq performed. Following clean up and QC, cells from the same mice were pooled together in the same ratios (CD45+:CD45-:Heps) as found in the tissue as a whole before sorting, different mice were then pooled together and the data were analyzed using scVI.(A–C) UMAPs showing annotations of cell types and proportions of each cell type as a % of total cells in the UMAP isolated using (A) ex vivo digestion; 13,144 cells, (B) in vivo digestion; 24,014 cells and (C) nuclei; 8,583 nuclei.(D) Average number of genes/cell in the annotated mac, B cell, hepatocyte, endothelial, and stromal cell populations following each isolation method. *p < 0.05 one-way ANOVA with Bonferroni post-test per cell type.(E) Correlation plot showing genes captured within the mac population when the liver is digested with the in vitro versus the in vivo digestion protocol.(F) Correlation plots showing genes captured within the mac, endothelial cell, and hepatocyte populations when cells are isolated using the in vivo digestion protocol or nuclei are isolated.(G–L) Confocal microscopy images to determine true abundance of (G) stromal cells and cholangiocytes (H) endothelial cells, (I) macs, (J) dendritic cells, (K) B cells, and (L) T cells in vivo. Scale bars, 200 µm.(M) The percentage of each population was calculated based on the percentage of a given population divided by the total number of nuclei. A threshold was applied to the DAPI channel (picture 1) in ImageJ (picture 2) and nuclei were automatically counted based on the ImageJ “analyze particles” plugin (size ). Due to the density of some liver zones, some nuclei were not automatically counted (arrow, picture 3). Those were then manually counted and added to the total number of nuclei. For the populations of interest, cells were counted manually based on specific markers (for example, CD3 for T cells, picture 4). Counting was performed blinded prior to analysis of the sequencing results.(N) Proportion of indicated cell types as a % of total cells identified in confocal microscopy images. Data are from 3–7 images per cell type taken from 2–4 mice.