Overexpression of Pitx2 induces morphological changes in Adult Liver Derived Progenitor Cells (ALDPC)

Recently, we could show that a subset of developmentally regulated fetal liver genes are also expressed during adult liver regeneration. One of these genes, Pitx2, was found to be strongly expressed in the early fetal liver at embryonic Day 9.5 p.c. as well as in a 2/3 hepatectomy model 24 hrs after resection. In the embryo, the Wnt signal pathway is important for the development of the organism, recently, there are also hints that this pathway plays a role in liver regeneration. Pitx2 is one of the genes regulated by Wnt signalling and recent hints indicate a role of this pathway also in liver regeneration. Via the overexpression of Pitx2 isoforms in ALDPC we set out to study the involvement in liver cell proliferation resp. maturation in more detail.Three isoforms of Pitx2 were overexpressed in ALDPC via lentiviral gene transfer with eGFP serving as marker gene. After three and seven days in culture, the amounts of eGFP positive cells were determined by FACS analysis. RNA samples were taken and the gene expression profiles were analysed with a RT- qPCR panel quantifying 42 liver specific transcripts.ALDPC are bipotential cells, which are able to differentiate–depending on culture conditions–either towards a hepatic or cholangiocytic phenotype. The Overexpression of the three isoforms Pitx2a, b and c leads to a microscopically visible change in phenotyp. The cells become bigger and have increased cytoplasma/ nucleus ratio compared to control cells. The analysis with the hepatic qPCR panel did not show any significant changes in most genes analysed. However, genes thought to be involved in cell-cell junction formation during epithel formation were upregulated (Claudin1 and Occludin) Detailed analyses of the expression studies and further investigations studying the involvement of Wnt-signalling will be presented and discussed.

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