Abstract
In the United Kingdom, the number of patients with liver failure awaiting
transplantation now exceeds the capacity of the nation's donor pool, with the
result that 20% of patients now die on the waiting list. The future for the
treatment of liver failure lies in part with cell-based therapies, in which liver
support may be provided on a short-term basis by biological liver-assist devices,
or in which sufficient mass of liver tissue may be transplanted into patients to
reverse liver failure as a graft. However, cell therapies are at a preliminary stage,
due to a lack of basic understanding as to how human liver cells can be made to
divide and to undergo functional maturation in vitro.
In order to understand how human liver cells can proliferate and differentiate in
vitro, a culture system was developed to support second trimester fetal liver
cells. Having characterised the culture system, and demonstrated viable
hepatocytes after seven days in vitro, experiments were carried out to determine
which circulating endocrine stimuli might initiate morphologic and functional
maturation in the developing hepatocytes. Cells were then incubated with
growth factors and cytokines and subject to two-colour flow cytometry to assess
which cell fraction might proliferate in vitro. Finally, urea metabolism and
protein secretion were assessed in the presence and absence of glucocorticoid
and different growth factors, to assess the interactions of these various stimuli at
a functional level.
The results showed that glucocorticoid alone brought about functional
maturation in terms of increased protein secretion, with significant increases
observed in a-fetoprotein, fibrinogen and a-i-antichymotrypsin secretion. This
represented increased secretion per cell, as there was no effect of glucocorticoid
on cell number. However, incubation with growth factors and cytokines showed
that EGF stimulated cellular proliferation. This proliferation occurred within a
primitive epithelial fraction, positive for cytokeratin 18, but negative for
fibrinogen. Final experiments showed that EGF and HGF had modest
stimulatory effects on urea synthesis. By contrast, KGF reduced urea synthesis
by channelling ammonia into anabolic pathways. With regard to protein
secretion, EGF inhibited fibrinogen and oc-i-antichymotrypsin secretion,
whereas, tumour necrosis factor inhibited fibrinogen alone. All of these
observations were made only in the presence of dexamethasone.
These data show that a satisfactory method for fetal liver cell culture was
developed. This model demonstrated that proliferation of liver epithelial cells
was stimulated by EGF, whereas functional maturation of fetal liver cells could
be brought about by exposure to glucocorticoid. Various growth factors and
cytokines had modest effects on urea and protein secretion, but only in the
presence of glucocorticoid. These experiments have provided new insights into
the maturational and proliferative signals in developing human liver. These data provide a frame of reference from which to develop cell-based therapies for the
treatment of liver failure in clinical practice.
