The origin and liver repopulating capacity of murine oval cells

Abstract

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.

Fig. 1.

Histology of DDC-treated liver. (A) Hematoxylin/eosin sample after 3 weeks. Arrows indicate the proliferating cells in the portal area. (B) BrdUrd staining (arrowheads) after 2 weeks of DDC treatment. (C and D) A6 antibody immunostaining (brown, arrows) at 3 (C) and 12 (D) weeks. A6 expression is seen in periportal small cells as well as some hepatocytes. (E and F) CK19 staining (arrows) at 3 (E) and 20 (F) weeks showing expansion of atypical duct cells. (G and H) Fah staining (arrowheads) at 3 (G) and 12 (H) weeks. The small periportal cells are Fah-negative.

Fig. 2.

Size profile of nonparenchymal cell fractions. (A) Normal liver. The peaks correspond to nonparenchymal cells (NPC) and hepatocytes (HC). (B) Liver after 3 weeks of DDC. There are three populations induced by DDC, which were the cells with diameters of 4, 7, and 10 μm. (C) F2 cell fraction of DDC-treated liver, consisting mostly of 10-μm cells.

Fig. 3.

Charazterization of the F2 cell fraction. F2 cells isolated by Nycodenz gradient centrifugation. (A-C) Immunocytochemistry of the same field. Blue stain is 4′,6-diamidino-2-phenylindole (DAPI) for nuclei. (A) albumin. (B) CK19. (C) albumin-CK19 overlay. (D) FACS analysis using antibodies to c-kit and CD45. About half (55%) of the cells were CD45-positive (hematopoietic in origin). The CD45-negative population could be divided into c-kit-positive and -negative cells.

Fig. 4.

Analysis of oval cells in chimeric livers. (A) Fah immunohistochemistry of a DDC-treated chimeric liver. Hepatocytes are stained dark (Fah-positive), whereas the periportal oval cells (arrow) are Fah-negative. (B) Southern blot using a probe to detect the Fah mutant (mut) and wild-type (Wt) alleles. DNA from the F2 and F3 Nycodenz cell fractions, hepatocytes (H), and unfractionated nonparenchymal cells (NPC) was analyzed. The nonparenchymal cells are predominantly Fah mutants, whereas hepatocytes are Fah wild-type. (C) Phosphoimager quantitation of Southern blot results shown in D. F2 = 97%, F3 = 81%, and H = 9% Fah mutant allele. (D) Semiquantitative PCR for the Fah genotypes. (Upper) Results from independently isolated F2 and F3 cells and hepatocytes (H). (Lower) Quantitation standards. F2 DNA contains >97% of the Fah mutant allele.

Fig. 5.

Liver repopulation by oval cells. (A) Competitive repopulation experiments. DNA from repopulated liver was probed with a neomycin-resistance gene fragment that detects different-sized fragments for the Fancc+/-, Rosa26+/-, and Fah-/- mutant strains of mice. Control DNA from the donor strains is shown in the two outermost lanes on each side. In the lanes marked 1:1, equal numbers of Fancc+/- hepatocytes and Rosa26+/- F2 fraction oval cells were cotransplanted. In the lanes marked 1:50, the ratio of hepatocytes to oval cells was 1:50. Hepatocytes and F2 cells contributed equally to repopulation when competed 1:1. All detectable repopulation was effected by the oval cells at the 1:50 ratio. (B) Fah immunohistochemistry shows oval cell-derived Fah+ hepatocyte nodules (arrows) in mutant liver after 8 weeks of transplantation.

Fig. 6.

Liver repopulation by oval cells from bone marrow chimeras. (A) Whole-mount β-galactosidase staining of liver from secondary recipients transplanted with liver cells from mice whose hematopoietic system had been replaced by lacZ-expressing cells (Rosa26+/-). The donor mice had been treated with DDC for 7 months. Controls for staining included wild-type (Wt) and Rosa-26 livers in the two left lanes. Controls for the transplantation included mice transplanted with CD45-/c-kit+ oval cells from a DDC-treated Rosa26+/- mouse (third lane from left). Dark staining (arrows) indicates areas of repopulation by lacZ-expressing hepatocytes. No blue staining was detectable in any secondary recipients of bone marrow chimeric mice, regardless of whether the donor cells were F2 cells or hepatocytes (far right lane). This result indicates that the oval cells and hepatocytes of the donor animals did not originate in the hematopoietic system. (B) Semiquantitative PCR of liver DNA aliquots from nine secondary recipients. Lanes 1-4 were from oval cell recipients, and lanes 5-9 were from hepatocyte recipients. The percentage of the Fah wild-type allele was 10-30% in all samples, whereas the Rosa-26 allele was not detectable in the same samples. The corresponding Fah enzyme activity measured is shown below each lane of Fah PCR (given in % of wild-type activity). Therefore, all observable liver repopulation was due to nonhematopoietic cells.

Fig. 7.

Model for liver stem cell hierarchy. In a chronic injury setting intrahepatic oval cell precursors produce oval cells, which can differentiate into either hepatocytes (HC) or bile duct epithelium (BDE). The reverse, i.e., dedifferentiation of hepatocytes to oval cells, does not occur. Hematopoietic stem cells (HSC) do not serve as precursors for oval cells, either directly or indirectly. Bone marrow-derived hepatocytes originate by cell fusion.