This document describes the use of 3D neurosphere assays to study developmental neurotoxicity. Neural stem/progenitor cells from fetal rat and human brains are cultured as neurospheres to model processes of brain development in vitro. The neurosphere assay can assess effects of compounds on proliferation, migration, differentiation and other pathways. It allows medium-throughput testing of compounds using automated imaging and analysis. Species-specific effects of compounds like valproic acid on rat and human neurospheres have been observed. The neurosphere assay is useful for safety testing and drug development by providing a 3D in vitro model of the developing brain.

1. NEUROSPHERES AS
3D IN VITRO MODELS
FOR ASSESSING DEVELOPMENTAL
NEUROTOXICITY
Prof. Ellen Fritsche
IUF- Leibniz Research Institute for Environmental Medicine

2. IUF Mission:
Molecular Preventive
Medicine
Düsseldorf, NRW, Germany
Gottfried Wilhelm Leibniz Scientific Community

3. Group of Sphere Models and Risk Assessment
Risk
Assessment
Molecular
Mechanism
Application
Safety
Testing
Drug
Development

4. The Neurosphere Method
Neural Stem/Progenitor
Cells (NPCs)
Fetal Brain
NPC Proliferation:
-Increase in size/time
-BrdU incorporation
Culture as Neurospheres
NPC Migration:
-Total migration distance
-number of migrated cells
-neuronal migration
NPC Differentiation:
-Neurons
-Astrocytes
-Oligodendrocytes

5. Proliferating Human Neurospheres
ß(III)Tubulin, Cx43, Nuclei

6. Migration
Moors et al. Environ Health Perspect 2009

7. Differentiating Human Neurospheres
ß(III)Tubulin, Cx43, Nuclei
Coop: Ole Klotz

8. Neurosphere Proliferation
HUMAN
DAY 0
DAY 7
RAT
DAY 0
HUMAN
DAY 7
RAT
Each sphere in a single well of a 96-well plate

9. Neurosphere Migration
HUMAN
HUMAN
RAT
RAT

10. Neurosphere Differentiation: Neurons
HUMAN
Hoechst ßIII-Tubulin
HUMAN
RAT
Hoechst ßIII-Tubulin
RAT

11. Neurosphere Differentiation: Oligodendrocytes
RAT
Oligodendrocytes (%)
Oligodendrocytes (%)
HUMAN

12. ß(III)tubulin/GFAP/DAPI
RAT
Synapsin/ß(III)tubulin/DAPI

13. The ‘Neurosphere-Assay’
Apoptosis
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
BrdU
Fritsche et al. Methods Mol Biol 2011
Gassmann et al. Toxicol in Vitro 2012
Bal-Price et al. ALTEX 2012
From Breier…Fritsche.. et al. Neurotoxicol Teratol 2009

14. The Neurosphere Method in Medium Throughput
Medium Throughput:
• Migration
• Neuronal Differentiation
• Neuronal Migration
Cellomics ArrayScan:
Nuclei and beta(III)tubulin
stainings of a differentiated
Human Neurosphere

15. 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’.

16. Acknowledgements
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
Cooperations:
High Content Imaging: Axel Mosig, University of Bochum
Proteomics: Prof. Kai Stühler, HHU Düsseldorf
LRI Innovative
Science Award 2006
German Alternative
Methods Award 2007
Leibniz DAAD
Thank you for your attention!