Transplanted Fibroblast Cell Sheets Promote Migration of Hepatic Progenitor Cells in the Incised Host Liver in Allogeneic Rat Model

Abstract

Introduction: ‘Cell-sheet engineering’ using temperature-responsive dishes has been noted as a new approach in the tissue engineering field. This technique has been already used in clinical fields including autologous mucosal epithelial cell sheets for the treatment of esophageal ulceration after endoscopic submucosal dissection. Usefulness of cell-sheet engineering has been expected also in hepatic tissue engineering. on the other hand, it was reported that proliferation of smooth muscle cells in the site where skeletal cell sheets were transplanted successfully prevented the deterioration of the impaired myocardium. These data indicate that transplanted cell sheets may induce organ-specific progenitor cells. The objective of this study is to explore procedure to induce hepatic progenitor cells in the rat liver. Methods: SD rat dermal fibroblasts (DFs) were used as donor cells and male F344 nude rats as recipients. DFs were cultured on temperature-responsive dishes and DFs were then harvested as a cell sheet. In the DF group, DFs sheets were transplanted into the incised surface of the liver. In the control group, only a same incision was made. The rats were sacrificed on postoperative days (POD) 7, 14, and 28 (n=5). for histological examination, hematoxyline-eosin staining and immunohistochemistry for cytokeratin (CK)-8 (rat bile duct epithelial cell marker), OV-6 (liver progenitor cell marker) and α-fetoprotein (AFP) (immature hepatocytemarker) were performed. We also performed the transplantation of GFP-expressing DFs sheet into the incised surface of the liver. Results: In the control group, necrosis or inflammatory cell infiltration was observed on POD7, and those were absorbed eventually. in contrast, the DF sheet transplanted sites became thicker, and bile duct (BD)-like structures appeared on POD 7. On POD 14, the BD-like structures were more significant, and immature hepatocyte-like cells were observed. Histological assessment revealed that BD-like structures were CK8 positive, and hepatocyte-like cells induced around the BD were both OV-6 and AFP positive. Observing the GFP-expressing DFs sheet transplanted sites byimmunofluorescent microscope, the proliferation of GFP positive DF cells were found, but the cells forming new structures including BD were GFP negative. These findings may become suggest that engrafted DFs recruited hepatic progenitor cells and supported their differentiation to mature hepatic tissues. Conclusions: We observed engineering process of hepatic tissues after transplantation of DF sheets. We also demonstrated that the origin of newly formed structures was recipient liver, which means transplanted DFs sheets induced hepatic progenitor cells from the recipient liver. The present study described a novel aspect of hepatic differentiation process induced at liver injury site. These technologies may become a useful therapeutic method for liver diseases in the future.