Hepatic progenitor cells, stem cells, and AFP expression in models of liver injury

Abstract

Adult hepatocytes and liver-cell progenitors play a role in restoring liver tissue after injury. For the study of progenitor cells in liver repair, experimental models included (a) surgical removal of liver tissue by partial hepatectomy; (b) acute injury by carbontetrachloride; (c) acute injury by d-galactosamine (GalN) and N-nitrosomorpholine (NNM); and (d) chemical hepatocarcinogenesis by feeding NNM in low and high doses. Serological and immunohistological detection of alpha-fetoprotein gene expression served to follow pathways of cellular differentiation. Stem cells were not required in models of surgical removal of parenchyma and in carbon tetrachloride intoxication of adult hepatocytes. In contrast, regeneration of liver occurred through biliary epithelial cells in injuries induced by GalN and NNM. These biliary epithelial cells, collectively called oval cells, are most probably derived from the canals of Hering. Proliferating bile duct cells reached a level of differentiation with reactivation of foetal genes and significant alpha-1-fetoprotein (AFP) synthesis signalling a certain degree of retrodifferentiation with potential stemness. Due to the same embryonic origin of bile ducts and hepatocytes, biliary epithelium and its proliferating progeny (oval cells) have a defined role in liver regeneration as a transit and amplification compartment. In their early proliferation stage, oval cells were heavily engaged in DNA synthesis ([3H]thymidine labelling). Pulse-chase experiments during experimental hepatocarcinogenesis exhibited their development into hepatocytes with high risk for transformation and leading to foci of altered hepatocytes. Hepatocellular carcinomas may arise either from proliferating/differentiating oval cells or from adult hepatocytes; both cell types have stem-like properties. AFP-positive and AFP-negative carcinomas occurred in the same liver. They may represent random clonal origin. The heterogeneity of phenotypic marker (AFP) correlated with a process of retrodifferentiation.

Figure 1

Regenerating mouse liver (BALB/cJ). Day 3 after partial hepatectomy; strong alpha-1-fetoprotein (AFP) immunoexpression in hepatocytes of portal areas (original magnification ×160).

Figure 2

Regenerating mouse liver (C3H/He), 3 days after carbon tetrachloride injury. Distribution of alpha-1-fetoprotein (AFP)-positive hepatocytes was comparable with that of injured BALB/cJ liver, but number of positive hepatocytes as well as their staining intensity was lower than in BALB/cJ mice. (a) Semithin Epon section (original magnification ×63). (b) Higher magnification view (original magnification ×250).

Figure 3

Localization of alpha-1-fetoprotein (AFP) in bile ductular cells (oval cells) of rat liver on day 3 after GalN injury. (a) AFP stained section counterstained with Mayer's haematoxylin, note AFP immunoreactive cells in ductular reaction areas in portal and in periportal area (original magnification ×160). (b) AFP-positive bile ductular cells (original magnification ×160).

Figure 4

Rat liver from day 28 during high dose NNM feeding, pulse labelling with [³H] thymidine. Thymidine incorporation in oval-shaped cells within the areas of ductular reaction (original magnification ×250, haematoxylin stain).

Figure 5

Rat liver from day 28–35 at high dose NNM feeding. (a) Proliferation of biliary epithelial cells and occurrence of numerous oval cells within portal and periportal area (original magnification ×160 haematoxylin & eosin stained section). (b) Histological section immunostained for alpha-1-fetoprotein (AFP), note AFP-positive oval-shaped cells which form biliary epithelial structures (original magnification ×160). (c) Higher magnification view of proliferated biliary epithelial cells which are stained for AFP and which form tubular structures with typical bile duct appearance (original magnification ×540). (d) High magnification view of [³H]thymidine labelled AFP-positive oval cells (original magnification ×540).

Figure 6

Rat liver from day 35 at high dose NNM feeding. Alpha-1-fetoprotein (AFP) immunoreactive ductular epithelial cells (oval cells) in transition towards hepatocytes giving rise to a nodule of hyperplastic appearance; note strong immunoexpression of AFP in both small oval cells and cells with the appearance of small intermediate-sized hepatocytes (original magnification ×160, haematoxylin counterstain).

Figure 7

Low magnification view of a hepatocellular carcinoma (original magnification ×16). (a) Section was immunostained for alpha-1-fetoprotein (AFP). (b) Serial section from same liver area with glycogen staining by periodic acid Schiff (PAS) reaction. Normal hepatocytes exhibit strong PAS reaction, while AFP-positive hepatocellular carcinoma remains PAS-negative.

Figure 8

Hepatocellular carcinoma; note alpha-1-fetoprotein (AFP)-positive carcinoma cells, adjacent normal liver is AFP-negative (original magnification ×160).

Figure 9

Schematic representation of normal liver structure and changes of its elements during disease and regeneration. (a) The lobular structure with the canal of Hering which drains bile from the bile canaliculi into the bile duct [modified from L.C. Junqueira & J. Carneiro (1980). In Basic Histology, Lange Medical Publications, p. 350]. (b) Oval cells can proliferate from the canals of Hering and lead to ductular epithelial structures. These proliferating cells are regarded as progenitor cells or intrahepatic stem cells which can differentiate via intermediate-sized hepatocytes into mature hepatocytes and bile duct epithelium; also, extra-hepatic stem cells of bone marrow origin are discussed to be a source of oval cells and provide progenitor cells for liver regeneration.