Download Generation of functional astrocytes from embryonic stem cells

Suppl. Linz / 2008
Poster: alternative testing methods for toxicity to reproduction
Generation of functional astrocytes from embryonic stem
cells for drug testing
Philipp Kuegler, Suzanne Kadereit, Bastian Zimmer, Marcel Leist
Doerenkamp-Zbinden Chair for in vitro alternative methods to animal experiments (Konstanz) (DE)
e-mail: [email protected]
Background motivation
Deleterious effects of neurotoxicants in the brain are not only caused as a result of direct neurotoxicity, but are also the result
of inflammatory processes caused by glial cells activated by the toxicant (Wyss-Corray et al., 2002). The main players
involved in inflammation in the brain are microglial cells and astrocytes (Falsig et al., 2006).
Embryonic stem cells (ESC) are a promising source for reliable and reproducible cell culture systems. Cells
generated from ESC have the potential to reduce the use of primary cells and possibly animal experiments. Recent advances
in the directed differentiation of murine ESC have led to robust methods for the derivation of neuronal cultures. However, no
method for the efficient generation of astrocytes from murine ESC has yet been published.
We are interested in developing methods that allow generation of cultures containing functional astrocytes. The
astrocytes are to be used to characterise toxicants.
Material and Methods
Mouse ESC (CGR8) were pre-differentiated and subsequently plated and further differentiated.
The astrocytes were characterised as to the expression of specific markers and compared to primary
astrocytes and astrocytes differentiated from a neural stem cell line. Furthermore, the astrocytes were functionally
characterised with regards to their inflammatory reactivity. Briefly, the astrocytes were incubated in the presence of
proinflammatory cytokines. Then, IL-6 and NO concentrations in the supernatants were measured.
Results
During the differentiation of both, embryonic and neural stem cells, astroglial markers (GFAP, S100b ,2
A2B5, CD44) were
upregulated while the neural marker Nestin as well as neuronal markers (NCAM, bIII-Tubulin) were downregulated. In the
stimulation experiments, we observed nuclear factor kB (NFkB) translocation into the nucleus upon stimulation as well as
an increased release of IL-6 and NO in the supernatant.
Discussion
The presence of astroglial markers (GFAP, S100b2
, A2B5, CD44) in about 80% of the cells of the culture and the absence of
the neuronal markers NCAM and bIII-Tubulin indicate a successful differentiation process.
Overall, mESC-derived astrocytes and neural stem cell derived astrocytes showed the same response pattern as
astrocytes isolated from mouse brain. The differentiation process can be affected by toxicants.
References
Falsig et al. (2006). J. Neurochemistry, 96(3), 893-907.
Wyss-Corray et al. (2002). Neuron 35(3), 419-32.
Keywords: astrocytes, embryonic stem cells, neurotoxicology
ALTEX 25, Supplement 1