Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation

Abstract

Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein.

Conclusion: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.

Figure 1. Mesenchymal−epithelial transition occurs in developing mouse liver.

(A) Phenotype changes of liver cells according to development stages. Representative images showed hematoxylin/eosin staining of livers from C57BL/6J mice at E11.5, 13.5 and 17.5 and 8 weeks (Adult) after birth. Right panels showed the magnified pictures. (B) Immunofluorescence for simultaneous detection of CK8/18 (epithelial) and vimentin (mesenchymal); (C) E-cadherin (epithelial) and N-cadherin (mesenchymal) in livers from mice in (A). Arrows in (B) showed the CK8/18 and vimentin overlapping cells. (D) The ratio for CK8/18 and/or vimentin expressing non-hematopoietic liver cells from mice in indicated development stages. Quantitative analyses were based on immunofluorescence staining. These images showed gain of epithelial characters and loss of mesenchymal characters within mouse liver development. Vim: vimentin. E: embryonic day. BV: Blood vessel. PV: Portal vein. Scale bars = 100 µm.

Figure 2. In vivo quantitative assay for mesenchymal−epithelial transition in liver developing of mouse.

(A) Relative mRNA expressions showed the increasing of Cdh1 (E-cadherin), CK18, and decreasing of vimentin, Snail1 and Twist1 at different developmental stages. RNA was extracted from non-hematopoietic liver cells of C57BL/6J mice. Error bars represented standard errors in three independent experiments. *P<0.05, **P<0.01. (B) Western blot for CK8/18 increasing and vimentin decreasing in non-hematopoietic liver cells of E11.5, 13.5 and adult mice. Actin was used as an internal control. E-cad: E-cadherin.

Figure 3. Isolation and characterization of hepatic stem cells of mouse.

(A) Hepatic stem cell sorting with flow cytometry. E13.5 mouse liver cells were sorted with gates set for c-kit⁻CD29⁺CD49f⁺CD45⁻TER119⁻ in order to isolate hepatic stem cells as mentioned in Materials and Methods. (B) Immunofluorescence for CK8/18 and vimentin expression in different isolated cell fractions with flow cytometry: CD45⁺Ter119⁺cells (hematopoietic cells), CD45⁻Ter119⁻cells (non-hematopoietic cells), c-Kit⁺CD49f^+/lowCD29⁺CD45⁻Ter119⁻ cells and c-Kit⁻CD49f^+/lowCD29⁺CD45⁻Ter119⁻ cells (hepatic stem cells). Samples were collected by cytospin. (C) Quantifications of CK8/18- and vimentin-expressing cell distributions in the different isolated cell fractions described in (B). Note that the CK8/18⁺vimentin⁺ co-expression cells were main population in stem cell fraction. Error bars represent standard errors in three independent experiments. Arrows showed the cell in magnified pictures. Scheffe's F test. *P<0.05, **P<0.01. Scale bars = 20 µm.

Figure 4. Mesenchymal−epithelial transition occurred in stem cell-derived colonies during culture.

Immunofluorescence for stem cells and stem cell-derived colonies at indicated culture days. (A) CK8/18 and vimentin transition accompanied with hepatic differentiation of stem cells into albumin positive cells; (B) E-cadherin and N-cadherin, vimentin transition accompanied with hepatic differentiation of stem cells. Scale bars = 100 µm.

Figure 5. In vitro quantitative assay for mesenchymal−epithelial transition of stem cell-derived colonies.

(A) Quantification of immunofluorescence in cultured stem cells on day 0 and stem cell derived colonies on days 7 and 21. Upper panel, E-cadherin and vimentin expressing cell assay (dashed regions indicate double-negative cells); lower panel, CK8/18, vimentin and albumin assay. Graph showed quantification of three independent experiments. (B) Relative mRNA expressions in cultured stem cells and stem cell derived colonies. It showed up-regulation of epithelial genes, Cdh1 (E-cadherin) and CK18, and down-regulation of mesenchymal genes, vimentin, Snail1 and Twist1 during stem cell differentiation according to the passing by of culture time. Error bars represent standard errors in three independent experiments. **P<0.01. Alb: albumin. E-cad: E-cadherin.

Figure 6. Vimentin-positive mesenchymal fetal liver cells are highly proliferative in vivo.

(A) Representative images of dual immunofluorescence of AFP and vimentin; (B) BrdU and vimentin in mice livers at different developmental stages. AFP and BrdU expressions decreased accompanied with vimentin reduction. (C) and (D) represented the relative quantitative assay of non-hematopoietic cells in (A) and (B) respectively. These results showed that AFP positive liver cells also expressed vimentin, and the vimentin⁺ cells are highly proliferative (BrdU⁺). Scale bars = 100 µm.

Figure 7. A Schematic model for MET.

MET is involved in stem cell inactivation, cell polarization and differentiation. This process is associated with a reduction of vimentin and accumulation of CK8/18 in stem cells. Furthermore, the variation of vimentin in stem cells is an indicator of cell proliferative activity. Conversely, it is suggested that EMT causes cells into active and de-differentiated state and acquire stem cell-like characteristics. M: mesenchymal state; E: epithelial state; Vim: vimentin.