File S1 - Usage of Adenovirus Expressing Thymidine Kinase Mediated Hepatocellular Damage for Enabling Mouse Liver Repopulation with Allogenic or Xenogenic Hepatocytes

Supporting figures. Figure S1. Hepatic identification of leukocyte, macrophages and neutrophils infiltration after AdTk/GCV mediated liver injury. The images represent liver sections of control mice, mice infected with AdTk alone (AdTk) or treated with GCV (AdTk + GCV). Images of mice treated with GCV alone are similar to the observed in control and AdTk mice. They show localization of leukocytes (CD45 antigen), macrophages and neutrophils in mouse parenchyma 4 weeks after GCV administration. Original magnification x20. Quantification of Kupffer cells and neutrophils were performed in three stained liver sections from 5 animals/time point/group using FIJI imaging software and obtained results were normalized with control group values. X axis represents the days post GCV administration and liver damage induction while Y axis represents normalized ratio between positive area and total area in arbitrary units. All AdTk+GCV groups present statistically significant differences compared to the Control and AdTk groups (*** pt-test). Figure S2. Analysis of oval cells, activated stellate cells and collagen deposition after AdTk/GCV mediated liver injury. Represent liver sections of mice without any treatment (Control), mice infected with recombinant adenovirus expressing HSV-Tk (AdTk) alone or infected with AdTk and treated with GCV (AdTk + GCV). Images obtained from mice treated with GCV alone livers are similar to the observed in control and AdTk mice. Murine oval cells were stained with A6 antibody and activated stellate cells with α-smooth muscle actin antibody 4 weeks after GCV administration (original magnification x40). Collagen deposition was demonstrated using picrosirius red staining. Original magnification x20. Figure S3. Presence of endothelial cells and bile canaliculi in GFP mouse hepatocyte nodules. Hepatic serial sections from a mouse transplanted for 14 weeks with murine GFP expressing hepatocytes were analyzed for (A) endothelial cells (mCD31) and (C) bile canaliculi (CD10). Hepatocytes derived from transplanted hepatocytes are identified as GFP positive cells (B). Original magnification x100. Figure S4. Quantification of human albumin concentration in human hepatocyte transplanted mice and analyzed for RI. (A) Peripheral blood was collected when animals were sacrificed and human albumin present in sera was quantified by specific human albumin ELISA analysis. The analyzed animals are the same used in the RI study (* pt-test). (B) Measurement of human albumin concentration in mice sera and human hepatocyte engraftment in individual mice. Figure S5. Identification of humanized regenerative nodules in the liver of mice transplanted with human hepatocytes. Serial liver sections from a mouse transplanted with human hepatocytes for 16 weeks after AdTk/GCV treatment were stained for hCK18 (A), HE (B) and hAlb (C) detection. Original magnification x100. Figure S6. Presence of endothelial cells and bile canaliculi in human hepatocyte nodules 4 weeks after transplantation. Hepatic serial sections from two mice transplanted for 4 weeks with human hepatocytes were haematoxylin-eosyn stained and analyzed for human hepatocytes (hCK18), endothelial cells (mCD31) and bile canaliculi (CD10). (A) And (B) represent hepatic sections from two different mice. Original magnification x200. Figure S7. HBV and HCV infection of mice transplanted with human hepatocytes. Mice transplanted with human hepatocytes for 8 weeks were inoculated with HBV human chronic carriers sera, animals were bled weekly and analyzed by PCR for the presence of HBV (A) and HCV (B) viral particles. In A, there are presented PCR results at week 5, 6, 7 and 9 after HBV inoculation and in B, there are presented PCR results at week 6, 7, 8, 9 and 10 after HCV inoculation. The numbers indicate animal identification, W is PCR reaction from non-infected animal sera and C+ is PCR positive control from patient sera used for inoculation. (PDF)