Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes

Abstract

Cell lines have many advantages: they can be manipulated genetically, expanded, and stockpiled for organ transplantation. Freshly isolated hepatocytes, oval cells, pancreatic cells, and hematopoietic stem cells have been shown to repopulate the damaged liver. Here we show that bipotential mouse embryonic liver (BMEL) stem cell lines participate in liver regeneration in albumin-urokinase plasminogen activator/severe combined immunodeficiency disease (Alb-uPA/SCID) transgenic mice. In the liver, BMEL-GFP cells proliferate and differentiate into both hepatocytes and bile ducts, forming small to large clusters detected throughout the 3-8 weeks analyzed after transplantation. Moreover, they respond like host cells to signals for growth, differentiation, and even zonal expression of metabolic enzymes, showing regulated expression of cytokeratins and liver-enriched transcription factors. Immunostaining for MHC class I molecules revealed that cells do not coexpress donor and recipient H-2 haplotypes, as would be the case had cell fusion occurred. This report shows that immortalized stem cell lines not only are competent to participate in the repair of a damaged tissue but also can differentiate into the two major epithelial cell types of a complex organ, hepatocytes and bile ducts.

Fig. 1.

BMEL cells integrate and proliferate in the liver of Alb-uPA/SCID mice without having undergone cell fusion and elicit an immune reaction. (A and B) Immunohistochemistry revealing BMEL-GFP cells (brown) in the hepatic parenchyma (A) and as a bile duct-like structure (B). (C and D) Clusters of BMEL-GFP cells (C) are composed of proliferating cells as shown by the nuclear localization of the Ki67 antigen (D). Broken lines in this and the following figures delimit the fields of interest. GFP (E), H-2K^k (F), and H-2D^d (G) immunohistochemical analyses of adjacent serial sections are shown. Transduced BMEL cells do not all express GFP (E). H-2K^k reveals all BMEL cells injected (F) and H-2D^d identifies the host cells (G). The H-2K^k-positive field (F) is H-2D^d-negative (G), demonstrating that cell fusion, which would result in double-positive cells, is not involved. (H) A complete liver section after GFP staining reconstituted from 31 photographic images and illustrating the material used for quantitation detailed in Table 1. BMEL cell clusters are visible as brown spots, whereas the purple-stained regions are necrotic. (I–N) Infiltrations of immune cells are seen around BMEL cells after transplantation. BMEL H-2K^k-positive bile ducts (J and M) are surrounded by CD45⁺ cells (I and L), of which many are macrophages (antibody F/480) (K) and neutrophils (antibody Nimp-R14) (N). (Scale bars: A and B,10 μm; C–G, 100 μm; H, 1 mm; I–N,40 μm.)

Fig. 2.

BMEL-derived hepatocytes and bile ducts localized within a portal triad show appropriate nuclear and cell-type-specific expression of liver-enriched transcription factors. BMEL-GFP cells are recognized as H-2D^d-negative (A), GFP-positive (C and E), or H-2K^k-positive (G). (B and D) HNF1α and HN4α, respectively, revealing nuclear staining of cells within the BMEL cluster of hepatocytes. (F) HNF4α is expressed in hepatocytes but not in bile ducts, and is absent from the BMEL-derived bile duct shown. (H) HNF1β is strongly expressed in bile ducts, including the duct formed by BMEL cells, whereas it is weakly expressed in hepatocytes. In B, D, F, and H, immune cells with small nuclei are visible surrounding the BMEL cell islands. BD, bile duct; A, artery; V, vein. (Scale bars: 100 μm.)

Fig. 3.

BMEL cells differentiate as hepatocytes and as bile ducts in the regenerating liver. (A–G) Analysis of serial sections with antibodies specificto hepatocyte markers showing that BMEL cells differentiate as hepatocytes in the liver, displaying the same characteristics as host hepatocytes. BMEL cells are recognized in A, C (GFP staining), and E (H-2D^d staining). All host and donor cells (A) in the section localize the bile canalicular marker DPPIV (B) to the apical pole. Note that clusters of large hepatocytes are seen in B for transplanted cells, and in C and D for host cells. Glutamine synthetase (D) is expressed by hepatocytes close to the central vein and by BMEL-GFP cells (C) located in this region. CPSI (F) is produced by hepatocytes throughout the liver parenchyma except those located in the central vein region, where glutamine synthetase (G) is expressed instead. The BMEL-derived hepatocytes (E) express these adult functions exactly as the neighboring host-derived hepatocytes. (H–M) BMEL-GFP cells differentiate as bile ducts in regenerating liver and express bile duct-specific CK7 and CK19. A bile duct formed by BMEL-GFP cells (H) expresses CK19 (I). A BMEL cell-derived bile duct recognized as H-2K^k-positive (J) expresses CK7 (K) as host-derived bile ducts. Regions of proliferating bile ducts are intrinsic to the Alb-uPA/SCID model because they are also observed in mice that have not been injected with BMEL cells. CK19 staining is restricted to bile ducts (L, GFP; M CK19). (Scale bar: 100 μm.)