Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures

Abstract

Organoid cultures of hepatocytes in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) display characteristic histologic organization. Biliary epithelium covers the surface of the tissue exposed to the culture medium. Hepatocytes, stellate cells and endothelial cells compose the underlying structures. In order to investigate the origin of the biliary epithelial cells in the organoid cultures, we utilized the retrorsine/DPPIV system of hepatocyte transplantation to create hybrid livers in which clones of DPPIV hepatocytes colonize variable portions of the lobules. We demonstrate that, as others have shown, biliary epithelium in this in vivo system remains that of the recipient (DPPIV negative) rat. Hepatocytes are the only cells positive for the DPPIV marker enzyme in the hybrid livers. Organoid cultures were prepared from the hybrid livers. Overall, 46.82% of the hepatocytes placed into culture were positive for DPPIV at time zero (after isolation). At 21 days in culture, 47.54% of the biliary epithelium on the surface of the organoid cultures was positive for DPPIV. Since the only DPPIV cells inoculated in the cultures were hepatocytes, this finding demonstrates that, in the conditions of the organoid cultures, hepatocytes do undergo phenotypic transition to biliary epithelial cells.

Fig. 1

(A) Histochemical stain for DPPIV of the hybrid livers at 3 months after infusion of DPPIV-positive hepatocytes into DPPIV-negative rat liver, after treatment with retrorsine and partial hepatectomy. Positive stain result is indicated by the canalicular pattern of red/orange color. Variable portions of lobules are occupied by the DPPIV-positive cells. (B) Higher-power photomicrograph of a portal triad from a hybrid liver. Many hepatocytes (H) are positive for DPPIV, whereas biliary epithelium (BD) is uniformly negative.

Fig. 2

DPPIV stain of air-dried smear of freshly isolated hepatocytes from a hybrid liver. Positive cells are stained red/orange. Normally the stain is limited to the bile canaliculus, but after collagenase perfusion, the canalicular structures are disrupted and canalicular markers are seen over the entire hepatocyte membrane.

Fig. 3

(A) Low-power (×20) and (B) high-power (×100) photomicrographs showing patches of DPPIV-positive biliary epithelial cells on the surface of the organoid structures at day 21. DPPIV-negative cells are interspersed between the DPPIV-positive cells, suggesting double origin of the biliary cells from both donor (+) and recipient (−) hepatocytes.

Fig. 4

DPPIV stain of organoid cultures at day 5. Several DPPIV-positive hepatocyte-like cells are seen near the surface of the cultures, but mature biliary epithelium is not yet present.

Fig. 5

Organoid cultures from hybrid livers at day 21. (A) Immuno-histochemical stain for cytokeratin 19, a marker for biliary epithelium. Most of the surface biliary epithelium is positive for cytokeratin 19. (B) Immunohistochemical stain for PCNA. Most of the surface biliary epithelium and the underlying hepatocytes have positive (brown color) nuclei, indicating the cells are into the cell cycle. (C) Immunohistochemical stain for the hepatocyte marker HEPPAR shows that the biliary epithelium (BD) is negative for this marker. A single hepatocyte (H) adjacent to the surface biliary epithelium is positive for HEPPAR.