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Non–cell autonomous effect of glia on motor neurons in an embryonic stem cell–based ALS model

Abstract

Here we report an in vitro model system for studying the molecular and cellular mechanisms that underlie the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Embryonic stem cells (ESCs) derived from mice carrying normal or mutant transgenic alleles of the human SOD1 gene were used to generate motor neurons by in vitro differentiation. These motor neurons could be maintained in long-term coculture either with additional cells that arose during differentiation or with primary glial cells. Motor neurons carrying either the nonpathological human SOD1 transgene or the mutant SOD1G93A allele showed neurodegenerative properties when cocultured with SOD1G93A glial cells. Thus, our studies demonstrate that glial cells carrying a human SOD1G93A mutation have a direct, non–cell autonomous effect on motor neuron survival. More generally, our results show that ESC-based models of disease provide a powerful tool for studying the mechanisms of neural degeneration. These phenotypes displayed in culture could provide cell-based assays for the identification of new ALS drugs.

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Figure 1: Derivation of Hb9::GFP;SOD1 mouse ESC lines.
Figure 2: Differentiation of the SOD1G93A mouse ESC lines into motor neurons.
Figure 3: Effect of SOD1 genotype on motor neuron survival.
Figure 4: Intracellular aggregation of SOD1 protein in cultured motor neurons.
Figure 5: Glial cell genotype directly affects motor neuron survival in culture.

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Acknowledgements

We thank J.W. Lichtman and J.R. Sanes for helpful discussions and use of confocal microscopy facilities at The Center for Brain Science, Harvard University. We are grateful to H. Akutsu for assistance in the derivation of mouse ESC lines, G. Birkhoff for preparing figures and B. Tilton for assistance with flow cytometry. We thank S. Przedborski and other members of the Columbia University research groups for communicating their results to us prior to publication, and for their criticals comments in our manuscripts. We thank Project ALS for supporting contact and cooperation between the research groups at Columbia and Harvard and for donating transgenic SOD1G93A mice. We thank T.M. Jessell and H. Wichterle for providing Hb9 antisera. This work was supported by the Stowers Medical Institute and Harvard Stem Cell Institute and US National Institutes of Health R01 HD046732-01A1 to K. Eggan and by the ALS Association to T. Maniatis. M.A. Carrasco is a Milton Safenowitz Post-Doctoral Fellow of the ALS Association and K. Eggan is a MacArthur Fellow of the John D. and Catherine T. MacArthur Foundation.

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Authors and Affiliations

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Contributions

F.P.D. derived the SOD1G93A, SOD1 and HB9::GFP mouse stem cell lines and performed both the differentiation of ESCs into motor neurons and the initial characterization of these neurons, including immunocytochemistry and initial neuronal counting experiments. M.A.C. performed morphometric analysis of SOD1 inclusions, ES-derived motor neuron coculture experiments with glia cells, and the associated characterization, survival assays and immunocytochemistry. M.S. contributed to the isolation of primary glia and glial genotyping. All authors contributed to experimental design and the writing of this paper.

Corresponding authors

Correspondence to Tom Maniatis or Kevin Eggan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Percentage of differentiating EBs cells that express GFP. (PDF 9308 kb)

Supplementary Fig. 2

Expression of neuronal marker Isl1 in cells derived from SOD1G93A mouse ES cell lines. (PDF 19109 kb)

Supplementary Fig. 3

Quantitative and qualitative analysis of SOD1 protein inclusions in ESC-derived motor neurons. (PDF 14249 kb)

Supplementary Fig. 4

Activation of caspase-3 and cytoplasmic localization of cytochrome C in SOD1G93A motor neurons 14 d after EBs dissociation. (PDF 14455 kb)

Supplementary Fig. 5

Characterization of primary glial monolayers derived from SOD1 and SOD1G93A mice. (PDF 14632 kb)

Supplementary Fig. 6

Table describes primary antibodies used. (PDF 16 kb)

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Di Giorgio, F., Carrasco, M., Siao, M. et al. Non–cell autonomous effect of glia on motor neurons in an embryonic stem cell–based ALS model. Nat Neurosci 10, 608–614 (2007). https://doi.org/10.1038/nn1885

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