| Authors |
Shi-Gang Xiong, Sharon Yavrom, Saswati Hazra, Da-Yu Wu, Hong-Yun She, Samuel W. French, Giuliano Ramadori, Hidekazu Tsukamoto; |
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| Abstract |
Background: Mesenchymal cells of the hepatic sinusoids (HSC) are thought
to represent the major source of excessive extracellular matrix in liver
fibrosis. The transdifferentiation of HSC to myofibroblasts (MF) is
believed to be a key event in hepatic fibrogenesis. However. HSC and MF
may still serve as two distinct cell types with the fibrogenic potential
in vivo, and it is not certain whether there is a transitional cell type
in liver fibrosis. We report here a HSC line, designated BSC, with the
phenotypes of both HSC and MF, which was established from HSC isolated
from rat experimental cholestatic liver fibrosis via spontaneous
immortalization.
Methods: HSC were isolated by sequential liver digestion with pronase and
collagenase followed by arabinogalactan gradient ultracentrifugation from
advanced liver fibrosis induced by 18-day cholestasis following bile duct
ligation in a male Wistar rat. The cells were collected from the interface
between the medium and a density of 1.034 and cultured originally in a 24
well plate in RPMI 1640 with 10% FBS and later in DMEM with 1g/L glucose
and 10% FBS. The cells were determined to be HSC with more than 95% purity
based on the typical morphology of freshly isolated MSC and UV-exited
fluorescence due to residual vitamin A storage. Unlike HSC isolated from a
sham-operated rat, the cells from a bile-duct ligated rat became
spontaneously immortalized and after 60 passages, clones were established
by the limiting dilution method. One of the clones, designated BSC-c10,
was characterized by immunofluorescent microscopy and Northern blot
analysis or RT-PCR for expression of HSC and MF markers at protein and
mRNA levels. Doubling time was assessed and 3H-proline and 3H-thymidine
were used for determination of collagen production and DNA synthesis in
response to TGFPI. Transient transfection with Lipofectamine 2000 (Gibco-
BRL) was tested.
Results: BSC has been maintained in culture for more than two years. A
doubling time is estimated to range 40-48 hr depending on culture
conditions. Most BSC clones maintain a polygonal shape of culture-
activated HSC when cultured in a low cell density, but their morphology
changes to a spindle shape of MF in a high density. BSC-c10 expresses: 1)
the markers known to be expressed by HSC such as glial acidic fibrillary
protein, synaptophysin, vascular cell adhesion molecule, neural cell
adhesion molecule; 2) those which are generally considered to be MF
markers, interleukin-6 and fibulin-2; and 3) those expressed by both cell
types, desmin, alpha(1)-procollagen, a-smooth muscle actin, and CD36. The
pattern of expression of these genes is not altered in a high density
despite the morphological change to the spindle shape. TGFbeta1 (1-10
ng/ml) causes a 40% decrease in DNA synthesis and a 33% increase in
collagen production in BSC-c10. The cells are easily transfected with a
GFP construct with as high as 60-70% transfection efficiency.
Conclusions: BSC-c10, one clone of the spontaneously immortalized HSC line
established from experimental biliary liver fibrosis, exhibits the
phenotypes of both HSC and MF, suggesting that a transitional cell type
may play a role in liver fibrosis. In vivo experiments need to be
performed to confirm this observation. Having phenotypic expression of
both cell types and high transfection efficiency, this cell line may be a
useful model for studying HSC transdifferentiation.
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