Study On Xenotransplantation Of Mouse STO Cell Lines Into Non-Immunosuppressed Adult Rat Livers

Objective: To investigate in vivo differentiation potential of a putative rat liver progenitor cell line, '3(8)#21-EGFP' cells were transplanted into DPPIV" German F344 rats.Methods: These rats produce DPPIV mRNAs of normal size (3.3-kb and 5.6-kb), but the translated DPPIV proteins are truncated and inactive. This defect renders recipient hepatocytes phenotypically DPPIV", but allows visualization of transplanted DPPIV+ donor cells against the DPPIV" background. Donor 3(8)#21-EGFP cells were retrovirally transduced to express EGFP and neomycin-resistance (neoR).Results: Initially, 3(8)#21-EGFP cells were reported to express properties of adult hepatocytes or bile duct cells dependant upon culture conditions, and they were assumed to be derived from syngeneic normal adult rat liver . During the first series of transplantation studies, however, we found that 3(8)#21-EGFP cells were derived from karyotypically abnormal embryonic mouse STO cells which had been ineffectively irradiated and used as feeder layers to isolate 3(8)#21-EGFP cells. Therefore, to eliminate immune privileges potentially conferred by y-irradiation, transduced retroviral DNA, and EGFP and neoR expression associated with such donor cells, unirradiated and unmanipulated parental STO cells were investigated as donors in a second series of xenotransplantation studies. Cells derived from embryonic mouse STO cell lines were transplanted intrasplenically into DPPIV" German Fischer rats. No immunosuppressive chemicals were administered. Donor cells moved rapidly into the liver as revealed by immunostaining. Four weeks later, the engrafted cells integrated into hepatic plates with -1% efficiency, and formed conjoint bile canaliculi as visualized by histochemical expression of donor cell-specific DPPIV activity and co-expression of hepatocyte-specific G-6-Pase and bile canalicular ATPase. Xenograft survival and differentiation were validated further between 1-12 weeks by findings of mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient livers, revealed by PCR and Plel restriction enzyme-coupled Southern blot-analyses, and nested RT-PCR and cDNA sequencing, respectively. Following donor cell transplantationinto DPPIV+ wildtype rats, mouse COX1 DNA was also detected intrahepatically by Southern blots 4-9 weeks later. Mouse DNA and mRNA molecules were not detected in livers derived from mock-transplanted animals or animals injected with donor cell DNA. Hepatocyte markers including DPPIV and G-6-Pase were detected by cytochemistry in small clusters of donor cells in confluent cultures; in contrast, mouse MHC class I H-2KQ , H-2DQ/LQ , class II I-A and rat MHC class I RT1A surface markers were not detected by immunofluorescent antibodies and flow cytometry, nor INF-r inducible.Conclusions: These observations indicate that under these experimental conditions, mouse STO cell lines are capable of xenoengraftment across major histocompatibility barriers, and differentiation along hepatocytic lineage.