The in vitro differentiating capacity of nonparenchymal epithelial cells derived from adult porcine livers

Abstract

Specific nonparenchymal epithelial cell (NPEC) clusters derived from normal adult porcine livers demonstrate a characteristic developmental pattern in the presence of other types of nonparenchymal cells in vitro. This pattern includes scattering, colonial growth, and an emergence of duct-like structures (DLSs) in the colonies. It has been confirmed that 96% of the scattered cell clusters in these cultures develop into colonies containing DLSs. In this study, we examine the differentiation of NPEC clusters using the scattered formation as a marker of the DLS-emerged colonies. We report that the NPECs expressed albumin, alpha-fetoprotein, transferrin, cytokeratin (CK) 18, CK7, and c-met, but not alpha-1-antitrypsin (AAT), at the scattering stage. In addition, at the same stage, NPECs expressed oval-cell-related markers such as OV6, but not biliary epithelial cell (BEC) markers such as gamma-glutamyltransferase, CK19, and CK14. At the DLS emerging stage, hepatocyte markers, including AAT, were detectable in the cells either at the periphery of colonies or in the cells surrounded by the DLSs. On the other hand, the cells constituting DLSs expressed BEC markers, suggesting a bile duct nature of the DLSs. Furthermore, the cells in the colonies possessed an ultrastructural appearance of differentiated hepatocytes and BECs. These results suggest that certain NPECs are bipotent, and that, in culture, they mimic hepatoblast development in vivo.

Figure 1.

Development of colonies derived from porcine nonparenchymal epithelial cell clusters. The cells within the same field marked by a needle are shown. A: Day 1 (28 hours after plating). The photograph within the square is at a higher magnification. NPECs are observed as clusters. B: Day 2 (50 hours). NPECs spread to about twice in area. C: Day 3 (81 hours). NPEC clusters begin to proliferate. D: Day 4 (101 hours). NPEC clusters begin to scatter. E: Day 4 (130 hours). F: Day 5 (141 hours). G: Day 7 (166 hours). H: Day 8 (197 hours). Arrows indicate a duct-like structure. I: Day 9 (220 hours). J: Day 10 (244 hours). K: Day 12 (292 hours). L: Day 15 (360 hours). Arrows indicate several duct-like structures. The cells, containing dark cytoplasm that surrounded by the duct-like structures, adopt a cobblestone-like morphology. The star indicates the position highly magnified in Figure 2 ▶ . Original magnifications, ×200 (A−C; scale bar, C, 50 μm) and ×100 (D−L; scale bar, L, 100 μm).

Figure 2.

High magnified microphotographs of Figure 1L ▶ . Cells surrounded by the duct-like structures are in focus (A) and those constituting duct-like structures are in focus (B). Original magnifications, ×400. Scale bar, 25 μm.

Figure 3.

Immunocytochemistry of scattered NPEC clusters on day 3 to 4. Cells show homogenous expression of each marker protein. Albumin (A) and transferrin (C) are expressed strongly in the cytoplasm and faintly in the nucleus. AFP expression (B) is detected in a granular pattern in the cytoplasm. CK7 (D), CK18 (E), OV6 (F), OC.10 (G), and vimentin (I) are present in a fibrous pattern in the cytoplasm. OV6 and vimentin are faintly detectable as compared to others. C-met (H) is detectable both in the nucleus and in the perinuclear region of the cytoplasm. All photographs are the same magnification. Scale bar (I), 50 μm. Insets show positive and negative (*) controls. a: HepG2. a*: NHDF. b: HepG2. b*: NHDF. f: Ac2F. f*: NHDF. i: CHO-K1. i*: HepG2. Original magnifications, ×200 (except insets, ×160). Scale bar, i*, 50 μm.

Figure 3.

Immunocytochemistry of scattered NPEC clusters on day 3 to 4. Cells show homogenous expression of each marker protein. Albumin (A) and transferrin (C) are expressed strongly in the cytoplasm and faintly in the nucleus. AFP expression (B) is detected in a granular pattern in the cytoplasm. CK7 (D), CK18 (E), OV6 (F), OC.10 (G), and vimentin (I) are present in a fibrous pattern in the cytoplasm. OV6 and vimentin are faintly detectable as compared to others. C-met (H) is detectable both in the nucleus and in the perinuclear region of the cytoplasm. All photographs are the same magnification. Scale bar (I), 50 μm. Insets show positive and negative (*) controls. a: HepG2. a*: NHDF. b: HepG2. b*: NHDF. f: Ac2F. f*: NHDF. i: CHO-K1. i*: HepG2. Original magnifications, ×200 (except insets, ×160). Scale bar, i*, 50 μm.

Figure 4.

Immunocytochemical analysis of duct-like structure-emerged colonies on Day 8 to 10. Albumin (A), transferrin (C), and AAT (D) are present in the cells at the periphery of colonies and in those surrounded by duct-like structures. AAT is detectable in the cells at the periphery of colonies as colonial spots. AFP (B) is expressed only in the cells at the periphery of colonies after Day 9. GGT (E), CK19 (F), CK14 (G), and OC.10 (L) are present in the cells constituting duct-like structures, although CK14 expression is faint. CK7 (H), CK18 (I), OV6 (J), and BD.2 (K) are expressed in all cells covering the duct-like structure-emerged colonies. Cells at the periphery of colonies express OV6 more strongly than those in the central parts of colonies. Original magnifications, ×100. Arrows indicate positive cells. Scale bar, L, 50 μm.

Figure 5.

Electron microscopy of cells surrounded by the duct-like structures and those constituting the structures. A: Some of the cells surrounded by the duct-like structures have numerous glycogen rosettes, tight junctions with desmosomes (arrows), and bile canaliculi-like structures with microvilli. B and C: Cells constituting duct-like structures show a large nucleus-cytoplasm ratio, a lumen structure with short microvilli (arrows in B), and juxtaluminal junctional complexes (arrow in C). The nuclei, which are notched in some cells, are located in the basal region of the cells. C: High magnification of the boxed area in B. Original magnifications, ×12,000 (A), ×1500 (B), ×6000 (C). Scale bars, 1 μm (A and C) and 5 μm (B).