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NEUROSPHERES AS 3D IN VITRO MODELS FOR ASSESSING DEVELOPMENTAL NEUROTOXICITY
Prof. Ellen Fritsche IUF- Leibniz Research Institute for Environmental Medicine
Gottfried Wilhelm Leibniz Scientific Community
Düsseldorf, NRW, Germany
IUF Mission: Molecular Preventive Medicine
Group of Sphere Models and Risk Assessment
Molecular Mechanism Application
Risk Assessment
Safety Testing
Drug Development
The Neurosphere Method
Fetal Brain Culture as Neurospheres
NPC Proliferation: -Increase in size/time -BrdU incorporation
NPC Differentiation: -Neurons -Astrocytes -Oligodendrocytes
NPC Migration: -Total migration distance -number of migrated cells -neuronal migration
Neural Stem/Progenitor Cells (NPCs)
Proliferating Human Neurospheres
ß(III)Tubulin, Cx43, Nuclei
Migration
Moors et al. Environ Health Perspect 2009
Differentiating Human Neurospheres
Coop: Ole Klotz ß(III)Tubulin, Cx43, Nuclei
Neurosphere Proliferation
HUMAN RAT
Each sphere in a single well of a 96-well plate
DAY 0 DAY 7 DAY 0 DAY 7
RAT HUMAN
Neurosphere Migration
RAT HUMAN
HUMAN RAT
Neurosphere Differentiation: Neurons
RAT HUMAN
Hoechst ßIII-Tubulin Hoechst ßIII-Tubulin
HUMAN RAT
Neurosphere Differentiation: Oligodendrocytes
RAT HUMAN
Olig
oden
droc
ytes
(%)
Olig
oden
droc
ytes
(%)
ß(III)tubulin/GFAP/DAPI Synapsin/ß(III)tubulin/DAPI RAT
From Breier…Fritsche.. et al. Neurotoxicol Teratol 2009
The ‘Neurosphere-Assay’
Apoptosis
BrdU
Fritsche et al. Environ Health Perspect 2005 Moors et al. Toxicol Appl Pharmacol 2007 Moors et al. Environ Health Perspect 2009 Moors et al. Genes & Immunity 2010 Tegenge et al. Cell. Mol. Life Sci. 2010 Schreiber et al. Environ Health Perspect 2010 Gassmann et al. Environ Health Perspect 2010 Verner et al. Toxicol in Vitro 2011 Fritsche et al. Methods Mol Biol 2011 Gassmann et al. Toxicol in Vitro 2012 Bal-Price et al. ALTEX 2012
The Neurosphere Method in Medium Throughput
Medium Throughput:
Cellomics ArrayScan: Nuclei and beta(III)tubulin stainings of a differentiated Human Neurosphere
• Migration • Neuronal Differentiation • Neuronal Migration
Summary & Conclusion
• Neurospheres of different species mimic processes of brain development in vitro.
• Application domains of neurospheres include safety testing and drug development.
• Species-specific VPA effects on rat and human neurospheres.
• Medium throughput is possible for compound testing in human and rodent neurospheres.
The 3D neurosphere assay can be used to study species-specific pathway-to-function effects (positive/negative) of compounds in a medium throughput fashion by
automated plating and usage of ‘High Content Image Analyses’.
Dr. Marta Barenys Dr. Kathrin Gassmann Dr. Julia Tigges Dr. Susanne Giersiefer Dr. Janette Schuwald Jenny Baumann Katharina Dach Martin Schmuck Maxi Hofrichter Katerina Kefalaki Stefan Masjosthusmann Thomas Temme Denise de Boer Ulrike Hübenthal
Thank you for your attention!
Cooperations: High Content Imaging: Axel Mosig, University of Bochum Proteomics: Prof. Kai Stühler, HHU Düsseldorf
Acknowledgements
LRI Innovative Science Award 2006 German Alternative
Methods Award 2007 Leibniz - DAAD