HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
radioimmunoassay. Initial experiments used methods described by Demetriou et al (1986a; 
1986b; 1986c) in which hepatocytes were grown on microcarrier beads and introduced into 
the peritoneal cavity. Low and inconsistent levels of human AAT were detected several 
days after transplantation, and microscopic examination of the transplanted material 
demonstrated that few carriers were vascularized (Ledley and Woo, unpublished data). 
The method reported by Thompson et al (1988) , in which hepatocytes are attached to Gel- 
foam™ soaked with angiogenesis factor and introduced into the peritoneum, also produced 
disappointingly low and short-lived expression of the transgene (Ponder and Woo, 
unpublished data). Significantly better results were obtained by injecting hepatocytes 
into the portal vein or spleen. Animals transplanted using these approaches expressed 
high levels of human AAT through the remainder of their lives (Ponder et al, 1991) 
(Appendix G) . 
Injection of hepatocytes into the spleen was found to be the preferred route for 
transplantation. Portal vein injection was associated with thrombosis, hepatic 
infarction, and death of the recipient in a fraction of animals and only 5 x 10^ cells 
could be successfully transplanted. Larger number of hepatocytes 2 x 10* could be 
transplanted into the spleen without complications, and higher levels of AAT expressed 
without any adverse effects were noted (Ponder et al, 1991). Similar experiments were 
performed using hepatocytes from a transgenic mouse expressing the E. coli E-gal gene 
under control of the liver specific AAT promoter, or hybrid mice expressing both E-gal 
and human AAT. Cells from these transgenic animals were identified by staining sections 
with X-gal which enabled identification of transplanted cells in the parench 3 mia of the 
liver after transplantation. These studies demonstrated that hepatocytes injected into 
the spleen could be recovered from the liver where they had assumed normal histological 
locations. No E-gal staining cells were identified in the spleen, and splenectomy 
performed several months after transplantation did not decrease the circulating levels 
of human AAT, suggesting that virtually all of the transplanted cells had migrated to 
the liver. No E-gal containing cells were identified in sections of other organs. 
Quantitative analysis of the number of X-gal staining cells in the transplanted liver 
and the levels of human AAT in the blood suggest that the nximber of cells expressing the 
transgene was equivalent to approximately 50% of transplanted cells. It is not known, 
however, whether this reflects engraftment of 50% of the Injected cells or engraftment 
of a smaller fraction with subsequent proliferation or overexpression of the transgene. 
Preliminary studies have demonstrated that the transplanted cells are capable of 
proliferation if partial hepatectomy is performed subsequent to transplantation 
(Darlington et al, unpublished data). More recent work has been directed at developing 
the surgical technologies for extending this work to larger animals. Several different 
approaches have been used including intraportal injection, intrasplenic injection, and 
injection into reservoirs (port-a-cath) inserted in the splenic vein. These preliminary 
experiments have demonstrated that it is possible to infuse >10® cells into these sites 
without surgical complications (Woo et al, unpublished data). 
I 'I 
Similar results have been reported from the laboratory of Dr. Roy Choudhary (Albert 
Einstein College of Medicine) who used hepatocytes expressing a recombinant hepatitis 
B surface antigen for analogous transplantation experiments (Gupta et al, 1990). 
Another animal model which has been the object of recent reports is the LDL- receptor 
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Recombinant DNA Research, Volume 14 
