Disappearance of GFP-Positive Hepatocytes Transplanted into the Liver of Syngeneic Wild-Type Rats Pretreated with Retrorsine

Abstract

Background and AimGreen fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP.MethodsHepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison.ResultsAll biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14.ConclusionGFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments using GFP and in interpreting their results.

Improving Hepatocyte Engraftment Following Hepatocyte Transplantation Using Repeated Reversible Portal Vein Embolization in Rats

“…More importantly, the transplanted hepatocytes were still visible at day 28, which was chosen for evaluation of hepatocyte engraftment in the present study. Interestingly, Maeda et al recently published results regarding the fate of GFP‐expressing hepatocytes transplanted in the liver of syngeneic wild‐type rats after retrorsine administration . Transplanted hepatocytes were still visible at day 28, but the authors demonstrated the disappearance of GFP‐expressing hepatocytes at day 42, most likely involving an immunological reaction against GFP.…”

Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments

“…Implying an underlying damaging process, GFP-positive hepatocytes in wild-type rats livers decreased more rapidly than the wild-type hepatocytes transplanted into the GFP transgenic rat livers. Moreover, GFP-positive hepatocytes attracted CD4+ and CD8+ infiltrating inflammatory cells, which is consistent with an immunological response [ 7 ]. Additionally, immunologic modification by bone marrow transplantation and administration of Tacrolimus, a T-cell inhibitor agent, showed increased survival of transplanted GFP-positive hepatocytes.…”

“…Hepatocytes obtained from GFP transgenic rats were transplanted into the livers of wild-type rats. For comparison, wild-type hepatocytes were transplanted into GFP-transgenic rat livers as well [ 7 ]. After showing engraftment using fluorescent microscopy, the transplanted hepatocytes were tracked for 48 days.…”

“…The folding and molecular structure (Fig. 1 ) both play integral roles in GFP’s fluorescent properties [ 7 ]. The formation of the functional chromophore in GFP, and that of analog proteins, entails three essential steps: protein folding, cyclization of the tripeptide chromophore motif, and oxidation of the cyclized chromophore [ 8 – 10 ].…”

Improving engraftment of hepatocyte transplantation using alpha-1 antitrypsin as an immune modulator

“…Previous studies have demonstrated that long-term hepatocyte proliferation and survival requires a combination of proliferative stimuli and suppression of host hepatocytes. This is typically done using partial hepatectomy which involves a 60–80% removal of the liver, and retrorsine treatment to inhibit endogenous hepatocytes [ 37 , 39 ]. The lack of liver pre-conditioning is one of the major limitations of this study.…”