Summary
(i) Provision of networks with mouse and human hepatocytes and characterization
Primary mouse hepatocytes (male B6 mice).
For the generation of high-quality quantitative data suitable for mathematical
modelling a standardized in vitro system is essential. Therefore, we have established
the "SOP-mouse-hep-quiescent" in our first funding period
(Klingmüller et al., 2006). This SOP allows isolation and cultivation of
quiescent (i.e. non-proliferating) hepatocytes from male Bl6 mice. These cells
are adequate for modelling of all signalling pathways within HepatoSys, including
JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-κB and Wnt/β-catenin.
We will supply the network with cells according to "SOP-mouse-hep-quiescent",
establish simple diagnostic assays that allow a rapid quality control and will
enforce that all network members use the same cells and SOP.
Hepatocytes according
to "SOP-mouse-hep-quiescent" do not proliferate (similar
to hepatocytes in healthy mouse livers that also show an only very low rate
of proliferation). However, one of the central milestones in HepatoSys II (especially
in the Network Regeneration) is the proliferating hepatocyte. Therefore, we
will establish clearly defined conditions that allow switching primary mouse
hepatocytes from a quiescent (according to "SOP-mouse-hep-quiescent")
to a proliferating state. These conditions will be defined in the new "SOP-mouse-hep-proliferating"
that will be established in cooperation with the Network Regeneration.
Specific culture
conditions are required to allow an optimal endocytosis in primary mouse hepatocytes.
We have already established conditions for "endocytosis competent hepatocytes"
that have been tested by the Network Endocytosis. These conditions will be further
optimized resulting in the "SOP-mouse-hep-endocytosis".
Characterization
of hepatocytes will include the transcriptome, proteome and enzyme activities.
Primary human hepatocytes isolated and cultured according to our "SOP-human-hep-quiescent" will be sent to groups of the Network Detoxification (1000 million cultured human hepatocytes per year), to the Network Endocytosis (200 million human hepatocytes per year) and to the Network Regeneration (500 million; only third year). Further information, in particular for possible clinical application of the isolated cells, will be gained from a database containing all relevant information of the donors.
(ii) Comparison of cultured hepatocytes with the intact liver
It should be guaranteed that modelling performed in vitro is relevant to the in vivo situation. Therefore, we will stimulate hepatocytes in vivo by injection of, for instance, HGF, IL6 or insulin into the portal vein. This procedure will allow comparing the dynamics of signalling in vivo and in vitro.
(iii) siRNA mediated knockdown - new SOPs
Key techniques required by several groups in HepatoSys are the siRNA-mediated knockdown of relevant signalling proteins and the transfection or transduction of primary hepatocytes (the latter techniques required to introduce siRNA as well as constructs of signalling factors). We will optimize transfection/transduction techniques in order to establish an SOP that allows the reproducible expression of vectors (including reproducible percentages of transfected hepatocytes, number of integrated copies and viability). The optimized technique will be applied to establish the siRNA-mediated knockdown of factors relevant for the Networks "Endocytosis" and "Regeneration".