Surface markers for the murine oval cell response

Abstract

The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated a new collection of monoclonal antibodies by immunization of Fischer rats with enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Each of the resulting antibodies recognized a surface antigen present on a liver cell subset and permitted the viable isolation of the associated subpopulation by fluorescence-activated cell sorting. Differential activity was observed on normal liver cells and at different stages of oval cell activation, indicating potential utility for progenitor cell identification. The subdivision of liver cells using these tools should facilitate the study of the biology of ductal and periductal hepatic cell types, including progenitors.

Conclusion: A new panel of surface reactive monoclonal antibodies to support investigation of the murine oval cell response has been developed.

Fig. 1

Antibody labeling of cell subsets in normal and DDC-treated mouse liver tissue. Mouse liver cryosections (5 μm) were labeled with supernatants from hybridoma cell lines with liver cell subset specificity. For each antibody, labeling on normal liver tissue (left) is compared to that of liver from a mouse fed a DDC-supplemented diet for 3 weeks (right). The secondary antibody was Cy3-conjugated anti-rat IgG adsorbed against mouse serum protein; nuclei were labeled with Hoechst 33342. Original magnification: 200×, inset: 1600×.

Fig. 2

Comparative labeling with the A6 oval cell marker on DDC-treated mouse liver tissue. Acetone-fixed cryosections were labeled with both A6 antibody (red) and the indicated antibody (green). Because each primary antibody is an unconjugated rat IgG, sequential labeling and a monovalent initial secondary antibody were employed. Nuclei were labeled with Hoechst 33342.

Fig. 3

Flow cytometric assessment of binding to viable dissociated mouse liver cells. Dead cells were excluded by gating for propidium iodide negative events; hematopoietic cells were excluded by gating for CD45⁻ events; and debris, cell clusters, erythrocytes, and hepatocytes were excluded by FSC/SSC gating. NPCs from normal and DDC-treated livers were sequentially labeled with the primary antibody and a PE-conjugated anti-rat IgG secondary antibody. The indicated gate for PE-positive events was set based on the negative control samples (A,B).

Fig. 4

Novel antibody labeling on liver NPCs is distinct from that of previously reported antibodies. (A,D,G) Negative controls indicate labeling with isotype control antibodies. Examples of comparative labeling with the antibodies described here and (B,C) Sca-1, (E,F) CD26/DPPIV, or (H) EpCAM are shown. EpCAM versus OC2-1D11 labeling is shown for (G,H) NPCs from untreated liver, whereas (A–F) the Sca-1 and CD26 comparisons used DDC-treated NPCs.

Fig. 5

A subset of antibodies binds hematopoietic cells. Bone marrow cells were collected from a mouse femur and labeled with the indicated antibodies. A small amount of nonspecific labeling was observed with (A) the secondary antibody alone or (B) with primary antibodies that do not bind marrow cells (for example, OC2-1C6). Plots in (C–E) illustrate positive labeling by the antibodies that recognize subsets of hematopoietic cells.

Fig. 6

Antibody labeling patterns change during oval cell activation. Cryosections of liver tissue obtained from mice after the indicated duration of DDC treatment were labeled with OC2 antibody supernatants (red) and rabbit anti-mouse CK19 (green). Secondary antibodies included Cy3-conjugated goat anti-rat IgG and Alexa488-conjugated donkey anti-rabbit IgG. Nuclei were labeled with Hoechst 33342. Original magnification: 200×, inset: 1600×.