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