Mesodermal mesenchymal cells give rise to myofibroblasts, but not epithelial cells, in mouse liver injury

Abstract

Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are believed to be the major source of myofibroblasts that participate in fibrogenesis by way of synthesis of proinflammatory cytokines and extracellular matrices. Previous lineage tracing studies using MesP1(Cre) and Rosa26lacZ(flox) mice demonstrated that MesP1+ mesoderm gives rise to mesothelial cells (MCs), which differentiate into HSCs and PFs during liver development. In contrast, several in vivo and in vitro studies reported that HSCs can differentiate into other cell types, including hepatocytes, cholangiocytes, and progenitor cell types known as oval cells, thereby acting as stem cells in the liver. To test whether HSCs give rise to epithelial cells in adult liver, we determined the hepatic lineages of HSCs and PFs using MesP1(Cre) and Rosa26mTmG(flox) mice. Genetic cell lineage tracing revealed that the MesP1+ mesoderm gives rise to MCs, HSCs, and PFs, but not to hepatocytes or cholangiocytes, in the adult liver. Upon carbon tetrachloride injection or bile duct ligation surgery-mediated liver injury, mesodermal mesenchymal cells, including HSCs and PFs, differentiate into myofibroblasts but not into hepatocytes or cholangiocytes. Furthermore, differentiation of the mesodermal mesenchymal cells into oval cells was not observed. These results indicate that HSCs are not sufficiently multipotent to produce hepatocytes, cholangiocytes, or oval cells by way of mesenchymal-epithelial transition in vivo.

Conclusion: Cell lineage tracing demonstrated that mesodermal mesenchymal cells including HSCs are the major source of myofibroblasts but do not differentiate into epithelial cell types such as hepatocytes, cholangiocytes, and oval cells.

Fig. 1

Contribution of MesP1+ mesoderm to HSCs, PFs, SMCs, and MCs, but not hepatocytes and cholangiocytes, in the adult liver. (A) A cell lineage analysis using the MesP1^Cre and R26T/G^flox mice. (B) Expression of TOMATO and GFP in the MesP1^Cre/+;R26T/G^f/f adult liver. Membrane-tagged TOMATO and GFP are observed in hepatocytes (hc) and HSCs (hsc) in the sinusoid, respectively. No GFP expression in hepatocytes (2,000 hepatocytes examined, n=3 mice). (C–I) Immunohistochemistry of the adult MesP1^Cre/+;R26T/G^f/f mouse liver for GFP with DES (C), PDPN (D), ELN (E), ACTA2 (F), CDH1 (G), HNF4 (H), or CK19 (I). Arrows indicate GFP+ HSCs in the sinusoid. Arrowheads and double arrows indicate DES+ ELN+ PFs adjacent to the bile duct (bd) and GFP+ MCs on the liver surface, respectively. Double arrowheads indicate DES+ ACTA2+ SMCs in the portal vein (pv) and hepatic artery (ha). GFP+ endothelial cells in the portal vein and hepatic artery are indicated by asterisks. No GFP expression in CDH+ HNF4+ hepatocytes and CDH+ CK19+ cholangiocytes (2,049 CDH1+, 1,836 HNF4+ hepatocytes and 1,036 CDH1+, 1,093 CK19+ cholangiocytes examined, n=3). Nuclei were counterstained with DAPI. Scale bar is 10 µm.

Fig. 2

In vitro activation of mesodermal HSCs. HSCs were isolated from the normal MesP1^Cre/+ or MesP1⁺⁺;R26T/G^f/f adult livers and were cultured for 6 (A) and 7 days (B–D). (A) Expression of TOMATO and GFP in HSCs. Asterisks indicate GFP+ HSCs isolated from the MesP1^Cre/+;R26T/G^f/f adult liver (Cre+). No GFP expression in HSCs from the MesP1⁺⁺;R26T/G^f/f liver (Cre−). (B) Immunostaining of GFP and ACTA2 or DES in HSCs isolated from the MesP1^Cre/+;R26T/G^f/f adult liver (Cre+). Asterisks indicate GFP+ HSCs expressing ACTA2 or DES. Arrows indicate GFP− HSCs expressing ACTA2 or DES. (C) A negative control without primary antibodies. (D) Immunostaining of GFP and DES. No GFP expression in HSCs isolated from the MesP1⁺⁺;R26T/G^f/f liver (Cre−). Nuclei were counterstained with DAPI.

Fig. 3

Similar phenotypes of GFP+ and GFP− HSCs in the MesP1^Cre/+;R26T/G^f/f liver. (A) HSCs were isolated from the MesP1^Cre/+;R26T/G^f/f (Cre+) or MesP1^+/+;R26T/G^f/f (Cre−) livers and were subjected to FACS. HSCs were analyzed by storage of VitA (FL5) and expression of GFP (FL1). (B) HSCs were isolated from the Cre+ and Cre− mice, cultured for 13 days, and subjected to FACS analysis. (C,D) HSCs were isolated from the MesP1^Cre/+;R26T/G^f/f liver and the primary (day 0) and cultured HSCs (day 13) were subjected to QPCR. In addition, VitA+GFP+ and VitA+GFP− HSCs sorted from the primary or day-13 HSCs were also subjected to QPCR for measurement for HSC markers (C) and other liver cell types (D). The results are expressed as relative expression compared against primary HSCs (day 0). The values were normalized against Gapdh. Each value is the mean ± standard deviation of the triplicate measurements.

Fig. 4

No contribution of mesodermal mesenchymal cells to hepatocytes and cholangiocytes in liver fibrosis. Lineage of mesodermal mesenchymal cells was traced using the MesP1^Cre/+;R26T/G^f/f mouse in liver fibrosis induced by CCl₄ injections 30 times (A–E) or by BDL for 2 weeks (F–J). (A,F) Expression of TOMATO and GFP in the liver. No GFP expression in TOMATO+ hepatocytes (hc) and cholangiocytes (bd). An arrow indicates GFP+ HSCs in the sinusoid. (B–E) Immunohistochemistry of the CCl₄-induced fibrotic livers with antibodies against GFP and ACTA2 (B), DES (C), HNF4 (D), or CK19 (E). GFP is expressed in ACTA2+ DES+ myofibroblasts (arrowheads) and DES+ activated HSCs in the sinusoid (arrows) but not in HNF4+ hepatocytes and CK19+ cholangiocytes (1,645 hepatocytes and 1,322 cholangiocytes examined, n=3 mice). (G-J) Immunohistochemistry of the BDL-induced fibrotic livers with antibodies against GFP and ACTA2 (G), DES (H), HNF4 (I), or CK19 (J). GFP is expressed in ACTA2+ DES+ myofibroblasts (arrowheads) and DES+ activated HSCs in the sinusoid (arrows) but not in HNF4+ hepatocytes and CK19+ cholangiocytes (1,852 hepatocytes and 2,560 cholangiocytes examined, n=3). Nuclei were counterstained with DAPI. Scale bars are 10 µm.

Fig. 5

No contribution of mesodermal mesenchymal cells to oval cells, hepatocytes and cholangiocytes in injured liver. Lineage of mesodermal mesenchymal cells was traced using the MesP1^Cre/+;R26T/G^f/f mouse in injured liver induced by DDC diet for 4 weeks. (A) Expression of TOMATO and GFP in the liver. No GFP expression in TOMATO+ oval cells (oc). Arrowheads indicate mesenchymal cells. bd, bile duct; ha, hepatic artery. (B–I) Immunohistochemistry of the injured liver with antibodies against GFP and A6 (B), EPCAM (C), PROM1 (D), CK19 (E), CDH1 (F), HNF4 (G), ACTA2 (H) or THY1 (I). (B–D) No GFP expression in oval cells expressing A6 antigen, EPCAM, and PROM1 (1,899 EPCAM+ oval cells examined, n=3 mice). Arrowheads indicate GFP+ myofibroblasts associated with GFP− oval cells. Asterisks indicate EPCAM+ oval cells. Double arrows show rare GFP+ cells embedded in oval cells. pv, portal vein. (E–G) No GFP expression in CK19+ cholangiocytes/oval cells and CDH1+ hepatocytes (hc)/oval cells, and HNF4+ hepatocytes (2,102 CK19+ cells, 2,749 CDH1+ cells and 2,120 HNF4+ hepatocytes examined, n=3). (H,I) Myofibroblasts associated with GFPcells, and HNF4+ hepatocytes (2,102 CK19+ cells, 2,749 CDH1+ cells and 2,120 HNF4+ hepatocytes examined, n=3). (H,I) Myofibroblasts associated with GFP− oval cells co-express GFP with ACTA2 or THY1. lv, lymphatic vessel. Nuclei were counterstained with DAPI. Scale bars are 10 µm.