Fig. 3: Liver repopulation with FAH-positive hepatocytes after in vivo LV-FAH delivery.

a Immunohistochemistry (IHC) for FAH in Nos. 166 and 167 at the time of euthanasia (337 days post-treatment) showing liver repopulation with FAH-positive hepatocytes in all areas of the liver. Sixteen areas of each liver were examined with similar results for a–c. b IHC for FAH in No. 162 at 60 days post-treatment showing multiple FAH-positive nodules in the liver with no presence of significant liver injury on H&E or Trichrome staining (first row). IHC for FAH from laparoscopic liver biopsy in No. 166 (second row) and No. 167 (third row) at 225 days post-treatment showing extensive liver repopulation with FAH-positive hepatocytes, with H&E and Trichrome staining showing hepatocellular inflammation with moderate fibrosis but no increased AFP staining. IHC for FAH in No. 166 (left medial lobe, fourth row) and No. 167 (left lateral lobe, fifth row) at 337 days post-treatment showing near complete liver repopulation with FAH-positive hepatocytes, with H&E and Trichrome staining showing resolution of hepatocellular inflammation and fibrosis with no increased AFP staining. c H&E and Trichrome staining of wild-type pig liver (left panels) and HCC in an NTBC under-treated FAH^−/− pig liver (No. 266, right panels) at 1 year show liver fibrosis and cellular alterations in the under-treated animal, while IHC for AFP shows ubiquitous increases in expression consistent with development of HCC in this context. d Quantification of AFP in No. 266 shows age-appropriate AFP levels in the neonatal period; however, AFP was slightly elevated at 1 year, while AFP in Nos. 166 and 167 at 337 days post-treatment was below the limit of quantitation.