Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

Abstract

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.

Endoglin Trafficking/Exosomal Targeting in Liver Cells Depends on N-Glycosylation

“…As a control, we also included sol-Eng in our initial experiments, because sol-Eng exosomal localization is already established [27]. Based on the fact that primary HSC and portal myofibroblast (pMF) are nearly inaccessible to standard transfection procedures [49], we constructed adenoviral constructs using the Ad-Easy-1 system for expression of FL-Eng and sol-Eng. These vectors are directed under the control of the ubiquitous CMV minimal promoter/enhancer driving effective expression of the 650 amino acid long FL-Eng (Figure 2) and the shortened 576 amino acid encoding soluble form lacking the transmembrane domain (TM) and the cytosolic domain (CD) comprising the whole extracellular domain in direct proximity to the established cleavage site (Figure 3, Supplementary Figure S2).…”

CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC

“…rat CFSC and human hepatic stellate cell line LX-2 (not shown). Using diverse chemically-enhanced transfection technologies we found that only approximately 6% of primary HSC and even a smaller fraction (< 1%) of fully transdifferentiated MFB are susceptible to gene transfer (Weiskirchen et al 2000). Therefore, we used in the following the immortalized rat cirrhotic fat storing cell line CFSC to test the impact of CCN3/NOV overexpression.…”

“…The cloning of Ad‐CMV‐NOV driving expression of full‐length rat NOV protein fused to a short C‐terminal 22‐amino acid peptide was recently described (Bohr et al 2010). Detailed protocols for amplification and purification of recombinant viral particles using CsCl gradient centrifugation and the BD Adeno‐X TM Purification Filter system (BD Biosciences, Clontech, Palo Alto, CA) were described before (Weiskirchen et al 2000; Nevzorova et al 2009).…”

Established Hepatic Stellate Cell Lines in Hepatology Research

“…There are also some general biochemical differences between continuous growing HSC lines and primary HSC. Exemplarily, it is well-accepted that primary HSC are hardly transfectable [27], while most of the lines are accessible for the uptake of foreign DNA. Similarly, continuous HSC lines can show divergent responsiveness towards growth factors.…”