Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts.
BACKGROUND
The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer.


RESULTS
With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively.


CONCLUSIONS
Our results indicate that FuGENE6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.
Weiskirchen
R
R
Institute of Clinical Chemistry and Pathobiochemistry, Central Laboratory, RWTH-University Hospital, Aachen, Germany. rweiskirchen@post.klinikum.rwth-aachen.de
Kneifel
J
J
Weiskirchen
S
S
van de Leur
E
E
Kunz
D
D
Gressner
A M
AM
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism* / Q000821:virology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D010969:Plastics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D010969:Plastics