Abstracts (poster) - Wissenschaft Online

Nadine Obier, Albrecht M. Müller
Analyzing epigenetic modifications in pluripotent stem cells
via intranuclear flow cytometry
Pluripotency describes the ability of embryonic stem cells (ES cells) to self-renew and to
give rise to all cell types of the developing embryo including the germline. Our goal is to
define pluripotency by identifying the entry point of cells into an irreversible
differentiation state with restricted differentiation potential.
In this regard we developed a FACS-based protocol which can quantitatively display
levels of different histone modifications in diverse cell types, such as in ES cells and in
their differentiated derivatives. Applying this intranuclear flow cytometric method, we
detected differences in global histone H4 acetylation levels between cells that were
either treated or not treated with the HDAC inhibitor TSA. Further, we observed a
distinct reduction of histone H3 lysine 27 tri-methylation (H3K27me3) levels in ES cells
lacking the protein EED, which – as a critical component of the Polycomb-group
repressor complex 2 - is participating in enzymatic methylation of H3K27. Our
preliminary studies on global levels of H3K4me3 and H3K27me3 in combination with ckit-surface
expression revealed that c-kit high immunophenotypic ES cells are also
highly positive for both H3K4m3 and H3K27me3 “chromatin-immunophenotypes”.
Together, we developed a new method for the analysis of global histone modifications by
intra-nuclear flow cytometry. This method represents a promising tool to simultaneously
study cellular properties, such as cell proliferation, apoptosis, surface marker expression
and intranuclear “chromatin-immunophenotype”, on the single cell level of large
quantities of cells.
contact:
M.Sc. Nadine Obier
Universität Würzburg
Institut für medizinische Strahlenkunde und Zellforschung (MSZ)
nadineobier@web.de
Versbacher Str. 5
97078 Würzburg (Germany)