Contribution of transplanted eGFP⁺ PSC to liver regeneration <i>in vivo</i>.

<p>(<b>A</b>) eGFP fluorescence (green) in PSC cultured for 7 days. (<b>B</b>) These culture-activated eGFP⁺ PSC were transplanted into wild type rats that underwent PHX in the presence of 2AAF. The eGFP⁺ PSC reached the host liver as determined by immunofluorescence staining of eGFP (green) 14 days after transplantation. (<b>C, D</b>) The eGFP⁺ PSC differentiated into hepatocytes in the host liver as determined by co-localization of eGFP fluorescence (green) and nuclear HNF4α immunofluorescence (red). (<b>E</b>) Their differentiation into hepatocytes was also confirmed by combined immunofluorescence staining of eGFP (green) and cytokeratin 18 (CK18; red). The fate of transplanted eGFP⁺ PSC was further analyzed by immunohistochemistry through Fast Red staining of eGFP-expressing cells (red). (<b>F, G</b>) The liver from wild type Wistar rats displayed no Fast Red staining after incubation with antibodies against eGFP, (<b>H</b>) but when eGFP⁺ PSC were transplanted, large areas of the host liver were colored red. This method labeled (<b>I</b>) hepatocytes (yellow arrows) and (<b>J, K</b>) bile duct cells (white arrows). (<b>J</b>) Other cell types in close proximity to bile ducts (black arrow) and liver sinusoids (yellow arrow) showed also eGFP expression as indicated by Fast Red staining. (<b>L</b>) The presence of eGFP-expressing cells in bile ducts of the host liver was further confirmed by combined immunofluorescence of eGFP (green) and CK19 (red). (<b>M</b>) Immunofluorescence staining of HNF4α (red) and FISH of chromosome Y (green) was used to identify hepatocytes in female liver tissue that derived from transplanted male PSC 14 days after PHX in the presence of 2AAF (white arrows). Also non-parenchymal cells with chromosome Y were detected (yellow arrows). (<b>N</b>) Combined FISH of chromosome Y (green) and immunofluorescence staining of panCK (red) revealed that the transplanted PSC had differentiated into duct-forming cholangiocytes. (<b>O, P</b>) Clonally expanded PSC of males were also transplanted into wild type female rats that underwent PHX in the presence of 2AAF. FISH of chromosome Y (green) and immunofluorescence staining of (<b>O</b>) HNF4α (red) or (<b>P</b>) panCK (red) indicated that single cell clones of PSC differentiated into hepatocytes and cholangiocytes (white arrows) as investigated after 14 days of regeneration. (<b>P</b>) Also PSC-derived cells that did not express cytokeratins were found close to bile ducts (yellow arrows). The cell nuclei were marked by DAPI (blue).