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DOI: 10.1055/s-0032-1332003
EpCam-specific Gold Nanoparticles for Detection, Live-imaging and Rapid Enrichment of Adult Human Liver Progenitor Cells
Hepatic stem cells and Hepatoblasts are the only two cell types known in the liver to express Epithelial Cell Adhesion molecule (EpCAM). We wish to exploit the specificity of this cell marker to:
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investigate how EpCAM-positive cells proliferate and emerge particularly in the first stages of tissue culture of primary human liver cell isolates
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observe how these immature cells are affected by other liver cell types in culture
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enrich and isolate liver progenitors from patient specific samples employing a fast sorting method as a replacement for our current „plate-and-wait“ method.
Laser-generated gold nanoparticles have been recently functionalized by various model biomolecules (peptides, oligonucleotides, antibodies, etc) by ultrashort-pulsed laser ablation in aqueous media, and showed major potential for biomedical applications.
For our first goal we plan to apply such highly pure laser-generated gold nanoparticles to stain EpCAM+ cells live and long term in human liver tissue samples. These nanoparticles will be directed to EpCAM+ cells and their visualization will be carried out by confocal microscopy, as gold nanoparticles provide long term live imaging without photobleaching and degradation in tissue culture.
For cell targeting, gold nanoparticles are planned to be conjugated with a commercially available monoclonal EpCAM antibody. Furthermore, it is known that membrane-bound EpCAM dimerizes with soluble EpCAM present in the supernatant. Thus, in an alternative approach, recombinant extracellular domain of EpCAM (EpEx), obtained from a mammalian expression system, will be conjugated to gold nanoparticles, replacing the sterically more problematic EpCAM-antibody.
In the initial steps we wish to show that the antibody-conjugated nanoparticles possess the same specificity as the antibodies on their own. For this we use progenitor cell populations recently isolated by our group, which express high levels of EpCAM in normal culture conditions but quickly loose this expression when treated with TGF-β under certain culture conditions, thereby differentiating to myofibroblasts-like cells.
Detailed data will be presented.
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