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 SOD1(G93A) allele showed neurodegenerative properties when cocultured with SOD1(G93A) glial cells. Thus, our studies demonstrate that glial cells carrying a human SOD1(G93A) 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.

Figure 1

Derivation of Hb9::GFP;SOD1 mouse ESC lines. (a,b) PCR for human SOD1 and the internal control Il2 (a), and for GFP in Hb9::GFP, Hb9::GFP;SOD1 and Hb9::GFP;SOD1G93A ESC lines (b). (c) Expression of human SOD1 protein in Hb9::GFP, Hb9::GFP;SOD1 and Hb9::GFP;SOD1G93A ESC lines. (d) A control Hb9::GFP transgenic embryo and an E10.5 chimera generated using the Hb9::GFP ESC line.

Figure 2

Differentiation of the SOD1G93A mouse ESC lines into motor neurons. (a) GFP expression of the ESC during different phases of the differentiation into motor neurons. (b–e) Expression of neuronal markers Tuj1 (b), Hb9 (c), Isl1 (d) and choline acetyltransferase (ChAT) (e) in motor neurons derived from SOD1G93A ESC lines at 9 and 14 d after the beginning of the differentiation process. (f) Presence of GFAP-positive cells surrounding a GFP motor neuron after 28 d. The white arrows indicate the nuclei of motor neurons immunoreactive for Hb9 or Isl1 proteins.

Figure 3

Effect of SOD1 genotype on motor neuron survival. (a) GFP-positive motor neurons derived from SOD1G93A and Hb9::GFP cell lines 61 d after differentiation. (b,c) Number of GFP-positive cells derived from Hb9::GFP (b) and SOD1G93A (c) ESC lines present 15, 30, 45 and 60 d after dissociation of embryoid bodies plated at two different concentrations (8 × 10⁵ and 4 × 10⁵ cells per well). (d,e) Number of GFP-positive motor neurons derived from Hb9::GFP and SOD1G93A 15 and 30 d after embryoid bodies dissociation plated at concentrations of 8 × 10⁵ (d) and 4 × 10⁵ (e) per well. (f,g) Same experiments analyzed as percentage of GFP-positive motor neurons derived from Hb9::GFP and SOD1G93A cell lines present at 15 d still remaining at 30 d after plating at 8 × 10⁵ (f) and 4 × 10⁵ (g) cells per well. Error bars represent s.d.

Figure 4

Intracellular aggregation of SOD1 protein in cultured motor neurons. (a,b) Expression of human SOD1 protein in motor neurons derived from SOD1G93A (a) and SOD1 (b) cell lines at 7, 14 and 21 d after embryoid body dissociation. (c) Co-expression of ubiquitin and SOD1 protein in cells derived from the SOD1G93A cell line, 28 d after differentiation. (d) Percentage of GFP-positive motor neurons with SOD1 inclusions present after 21 d in culture.

Figure 5

Glial cell genotype directly affects motor neuron survival in culture. (a) GFP- positive motor neurons at 5, 7, 14, 21 and 28 d in culture after embryoid body dissociation. (b) Graph shows percentage of Hb9::GFP– positive cells over time in all the conditions studied. Experiments were done in triplicate and results were normalized to the number of cells found at 7 d in vitro. Error bars represent s.e.m.