Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice

Abstract

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

Figure 1

Histology of normal and injured liver. (A): Normal liver, no BMT. (B): No liver injury, 12 weeks after BMT. (C): Liver irradiation, 7 days after injury. Arrows indicate mitotic hepatocytes. (D): DDC, 7 days. (E): CCl₄, 2 days after a single administration. Arrow indicates necrotic pericentral hepatocytes. (F): CCl₄, 14 days after a single administration. Arrow indicates ductular proliferation. Abbreviations: BMT, bone marrow transplantation; CCl₄, carbon tetrachloride; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

Figure 2

Immunofluorescent localization of GFP. (A): GFP is expressed at high levels in approximately half of hepatocytes (arrow) in the beta actin-GFP mice used as bone marrow donors. GFP is also variably expressed in bile duct cells (arrowhead). (B): Nontransgenic control liver. Note background autofluorescence in hepatocytes. (C): Nontransgenic recipient of GFP donor BMT, with GFP detection by DAB immunohistochemistry. A potential marrow-derived hepatocyte (enlarged in inset) stains brown. (D): Nontransgenic recipient of GFP donor BMT, with GFP detection by immunofluorescence (green). Numerous GFP-positive cells are seen surrounding a bile duct and passing through sinusoids. (E): Colocalization of Y chromosome (small pink nuclear dot) and GFP. Both are markers of male GFP+ donor origin in this nontransgenic female recipient liver. A sinusoidal cell staining for GFP but not Y-FISH is indicated by the arrow. A cell staining for Y chromosome but not GFP is indicated by the arrowhead. A Y-positive potential marrow-derived hepatocyte is circled in red. The large orange cells are autofluorescent erythrocytes. Abbreviations: BMT, blood marrow transplantation; DAB, 3,3′-diaminobenzidine; GFP, green fluorescent protein; Y-FISH, Y chromosome fluorescence in situ hybridization.

Figure 3

Costaining of recipient female liver after male BMT and liver injury with CCl₄. Y-FISH (small pink nuclear dot) and CD45 + F4/80 (combined to highlight leukocytes, green). (A): Liver section at ×40 magnification. White boxes indicate magnified views in (B) and (C). (B): Top right: magnified view: Note Y+ (pink dot) and CD45 + F4/80-positive (green) cells in the top left, and a Y+ potential hepatocyte (circled in white). (C): Bottom left: Numerous Y-positive cells near the bile duct and portal vein do not stain with CD45 + F4/80 (arrowhead in [C]), indicating that the absence of CD45 + F4/80 staining is not sufficient evidence of epithelial phenotype. Abbreviations: BMT, bone marrow transplantation; CCl₄, carbon tetrachloride; Y-FISH, Y chromosome fluorescence in situ hybridization.

Figure 4

Colocalization of Y chromosome (pink) and cytokeratin (green). Pancytokeratin immunofluorescence stains cholangiocytes and ductal progenitor cells strongly, but stains hepatocytes weakly. (A): Male liver. (B, C): Female recipient of male BMT followed by liver injury with DDC. Note that cytokeratin-positive oval cells are surrounded by donor-derived (likely inflammatory) cells. Arrow in (C) indicates a potential marrow-derived cholangiocyte. Abbreviations: BMT, bone marrow transplantation; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

Figure 5

Colocalization of Y chromosome (pink) and HNF1 (green nuclear stain). (A): Male control liver. (B): Female control liver. (C): Female FAH null recipient after male donor BMT and NTBC withdrawal to select marrow-derived hepatocytes. (D–F): Female recipient of male BMT, followed by DDC treatment. (D): Y-positive, HNF1-negative cells (arrowheads) are donor-derived blood cells, clearly distinguished from native hepatocytes (HNF1-positive, Y-negative). (E): Y-negative, HNF1+ hepatocytes (short arrow) and cholangiocytes (long arrow) can be distinguished from Y+, HNF1-negative cells (arrowheads) in DDC-treated female mice after male donor BMT. (F): Inflammatory cells infiltrating a bile duct are Y-positive, HNF1-negative. DDC-induced cholestasis causes hepatocyte cytoplasm to appear reddish-orange in (E) and (F). Abbreviations: BMT, bone marrow transplantation; DDC, 3,5-diethoxycarbonyl-1,4-dihy-drocollidine; FAH, fumaryl acetoacetate hydrolase; HNF1, hepatocyte nuclear factor-1; NTBC, 2-(2-nitro-4-trifluoro-methyl-benzyol)-1,3-cyclohexanedione.

Figure 6

Colocalization of Y chromosome (pink nuclear dot) and HNF1 (green nuclear stain) in female recipients of male BMT, after liver injury. (A): Y-positive, HNF1-positive hepatocyte nucleus (arrow) in a mouse treated with two doses of CCl₄ over 7 days. (B): Y-positive, HNF1-positive hepatocyte nucleus (arrow) in a mouse treated with eight doses of CCl₄ over 8 weeks. (C): Y-positive, HNF1-positive hepatocyte nucleus (arrow) in a mouse treated with DDC for 10 days and allowed to recover for 7 days. (D): Confocal image of the hepatocyte in (C) demonstrates localization of Y chromosome and HNF1 in the same plane as nuclear DNA (DAPI). Abbreviations: CCl₄, carbon tetrachloride; DAPI, 4′,6-diamidino-2-phenylindole; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; HNF1, hepatocyte nuclear factor-1.