Hepatic progenitor cells in human liver cirrhosis: immunohistochemical, electron microscopic and immunofluorencence confocal microscopic findings

Abstract

Aim: To investigate whether hepatic progenitor cells (HPC), that reveal the features of oval cells in rodents and small epithelial cells (SEC) in certain human liver disease, were also found in human liver cirrhosis (HLC).

Methods: Surgical liver specimens from 20 cases of hepatitis B virus-positive HLC (15 cases containing hepatocellular carcinoma) were investigated by light microscopic immunohistochemistry (LM-IHC). Among them specimens from 15 cases were investigated by electron microscopy (EM) and those from 5 cases by immunofluorencence confocal laser scanning microscopy (ICLSM). Antibodies against cytokeratin 7 and albumin were used and single and/or double labelling were performed respectively.

Results: LM-IHC showed that at the margins of regenerating nodules and in the fibrous septae, a small number of cells in the proliferating bile ductules were positive for CK7 and albumin. At the EM level these HPC were morphologically similar to the SEC described previously, and also similar to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size, oval shape, a high nucleus/cytoplasm ratio, a low organelle content in cytoplasm, and existence of tonofilaments and intercellular junctions. ICLSM revealed that HPC expressed both cytokeratin 7 and albumin.

Conclusion: HPC with ultrastructural and immunophenotypical features of oval cells, i.e., hepatic stem cell-like cells as noted in other liver diseases, were found in HLC. These findings further support the hypothesis that bipotent hepatic stem cells, that may give rise to biliary epithelial cells and hepatocytes, exist in human livers.

Figure 1

HLC. Several proliferating bile ductules are seen at the edge of a regenerating nodule. There is a dense, mostly lym-phocytic inflammatory infiltrate. Haematoxylin and eosin × 200.

Figure 2

HLC. Immunohistochemical staining shows that cells of proliferated bile ductules are strongly positive for CK7. Anti-CK7, × 200.

Figure 3

HLC. Some small hepatic progenitor cells, immu-noreactive with albumin are seen at the edge of regenerated nodule. Anti-albumin × 200.

Figure 4

HLC. Double labelling immunohistochemistry shows that cells of proliferated bile ductules are positive for CK7 (brown), hepatocytes are positive for albumin (green) and a few HPC are stained with both CK7 and albumin (brown-green). Anti-albumin and CK7, × 200.

Figure 5

HLC. Under electron microscopy, two HPC charac-terized by small size (approximately 10 μm), oval shape, and high nucleo/cytoplasm ratio, are seen. They are adjacent to hepatocytes (H) on one side and to HPC on the other side. × 5 000.

Figure 6

HLC. At higher magnification, cytoplasm of HPC is seen to contain tonofilaments (thin arrow) and intercellular junctions (thick arrows) between HPC and hepatocytes. × 16 000.

Figure 7

HLC. Immunofluorencence confocal laser scanning microscopy (ICLSM) indicates at edge of regenerating nodules, proliferating bile ductules in which not only bile epithelia cells (anit CK7, green), but also HPC (anti CK7 and albumin, orange) are noted. × 1 800.

Figure 8

HLC. Immunofluorencence confocal laser scanning microscopy (ICLSM) shows that two proliferating bile ductules are composed only of bile epithelial cells (anit CK7, green), but one proliferating bile ductule contains HPC (anti CK7 and albumin, orange). × 1 000.