Figure 2. Disappearance of GFP-positive hepatocytes in wild-type syngeneic rat liver.

(A) Timeline of Experiment 1. Recipient rats received retrorsine treatment twice (black arrow). One week after hepatocyte transplantation (large black triangle), all rats had a liver biopsy under general anesthesia, and then rats were sacrificed on days 14, 28, or 42 (white triangle). (B) Confocal microscopic observation of GFP fluorescence in hepatocytes after transplantation into wild-type Lewis rats. (Upper lane) The liver biopsy taken at day seven shows engraftment of transplanted hepatocytes in all cases. The nodules of GFP-positive hepatocytes after transplantation were less numerous at day 28 than at day 14. GFP-positive hepatocytes at 28 days also showed more heterogeneous GFP expression with an irregular cell shape than at day 14, indicating cytoplasmic degenerative changes. The dissociation of GFP-positive hepatocytes was apparent along with nuclear debris (small green dots). The infiltration of GFP-negative cells with a small nucleus (blue stain) was also apparent. (Lower lane) The progressive growth of GFP-negative hepatocytes in GFP-Tg Lewis rat liver resulted in the size of each cluster of transplanted hepatocytes increasing with time. (C) Higher magnification of the liver tissue at 28 days after hepatocyte transplantation from GFP-Tg rats to wild-type rats. (D) Merged image of immunofluorescent staining for albumin (red) and immunohistochemical staining for GFP (green). The transplanted hepatocytes from wild-type rats proliferate and express albumin in GFP-Tg Lewis rat liver. (E)The percentage of transplanted GFP-positive hepatocytes increased by 14 days after cell transplantation, but significantly decreased by 28 days, and no GFP-positive hepatocytes were observed at day 42. In contrast, the percentage of GFP-negative transplanted hepatocytes increased steadily. (F) PCR analysis of the GFP transgene showed a reduction at 28 and 42 days, suggesting elimination of the GFP transgene.