Cell networks in the mouse liver during partial hepatectomy

Abstract

In solid tissues, homeostasis and post-injury regeneration involve a complex interplay among various cell types. The mammalian liver harbors numerous epithelial and non-epithelial cells, and the global signaling networks governing their interactions are unknown. To unravel the hepatic cell network, we purified 10 different cell populations from normal and regenerative mouse livers. Analyzing their transcriptomes unveiled ligand-receptor interactions and over 50,000 potential cell-cell interactions in both ground state and after partial hepatectomy. Importantly, about half of these differed between the two states, indicating massive changes in the cell network during regeneration. Our study provides the first comprehensive database of potential cell-cell interactions in liver cell homeostasis and regeneration. Leveraging this predictive model, we identified and validated two previously unknown signaling interactions involved in accelerating and delaying liver regeneration. Overall, we provide a novel platform for investigating autocrine/paracrine pathways in tissue regeneration, with broader applications to other complex multicellular systems.

Keywords: FACS; Fstl1; Sfrp1; liver regeneration; partial hepatectomy; stem cell.

Graphical abstract

Figure 1

Cell-cell interactions in homeostatic and regenerative liver (A and B) Venn diagrams of the number of paracrine and autocrine interactions in normal liver and PHx among all 10 cell types (A) and hepatocytes only (B). (A) There are 19,528 unique interactions in normal liver and 23,640 in PHx. The normal and PHx states had 39,630 shared common interactions. (B) In hepatocytes, there were 1,632 unique interactions in normal and 1,671 after PHx, with 3,342 shared common interactions. (C and D) Schematic top 10 CCInx-weighed interactions in normal (C) and PHx (D) livers by Cytoscape network visualization. The interactions are ranked by the edge weights calculated by CCInx. PHx, 24 h after 70% PHx; HC, hepatocyte; blood: blood lineage cells; Thy1, Thy1+ cells. Arrows indicate either autocrine (from one cell to itself) or paracrine (from one cell to another) interactions. See also Figures S1 and S2.

Figure 2

Fstl1 overexpression promotes hepatocyte proliferation (A and B) Cytoscape network visualization of Fstl1 as a ligand in 10 different cell types from the normal (A) and PHx (B) livers. Arrows indicate the paracrine and autocrine signaling pathways. Red arrow indicates that the paracrine signaling pathway appears in the PHx but not in the normal liver. (C and D) The CCInx database showed Fstl1 as a ligand in LSEC and its top 10 receptors in HC. Fstl1 is off (yellow circle) (C) in the normal liver but upregulated and on (dark purple circle) after PHx (D). (E) Normalized gene expression of Fstl1 (tag counts in FPKM) in 10 different cell types from normal liver and PHx. Tag counts in red color, the Fstl1 gene is off; tag counts in green color, the Fstl1 gene is on. (F) rAAV construct for overexpressing the Fstl1 gene. The Fstl1 gene was cloned into an AAV2 vector backbone with the human thyroid hormone-binding globulin (TBG) as promoter. 2 × 10¹¹ vector genomes of AAV-Fstl1 virus were injected into C57BL/6 mice fed with BrdU in the drinking water. The liver was harvested on day 7 after injection. (G) Representative morphology of mouse liver after Fstl1 overexpression. A liver transduced with an AAVDJ-Tomato vector was used as control. (H) Liver-body weight ratio of independent mice treated with AAVDJ-Tomato (n = 9) and AAVDJ-Fstl1 (n = 15). Student’s t test. p > 0.05. (I) Histology of anti-BrdU staining in the control (Tomato) and Fstl1 overexpression liver. Scale bar, 50 μm. BrdU-expressing nuclei stained brown. (J) Percentage of BrdU+ hepatocytes from Fstl1 overexpression (n = 10) and mock (Tomato) (n = 5) transduced mouse liver. Student’s t test. ^∗p = 0.007. HC: hepatocytes. See also Figure S3.

Figure 3

Sfrp1 delays liver regeneration (A and B) Sfrp1 pathway in 10 different cells in the normal liver (A) and 24 h after 70% PHx (B). Arrows indicate the paracrine and autocrine signaling pathways. (C and D) Sfrp1 as a ligand in Thy1+ cells and its receptor Fzd6 in NPD. (E) FPKM mapped reads in 10 different cell types from normal liver and PHx. Tag counts in red color, the Sfrp1 gene is off; tag counts in green color, the Sfrp1 gene is on. (F) AAV8-Sfrp1 was transfected into a wild-type mouse 7 days before feeding with BrdU drinking water. One day after BrdU water treatment, PHx was performed. Liver harvest was carried out at 36, 48, 72, and 240 h (day 10) after PHx. (G) Representative morphology of mouse liver overexpressing Sfrp1 with PHx at 36, 48, 72, and 240 h (day10). Liver transfected with AAV8-GFP was used as control. (H) Histology of anti-BrdU staining in the mouse liver treated control (AAV8-GFP) and Sfrp1-overexpressed mouse liver at different time points. Scale bar, 50 μm. BrdU-expressing nuclei stained brown. (I) Quantitative assay of the percentage of BrdU⁺ hepatocytes in the mouse liver treated with control (AAV8-GFP, n = 3) and AAV8-Sfrp1(n = 5) at different time points. Statistical analyses: Mann-Whitney U test. ^∗∗p < 0.01. See also Figure S3B.