The capacity of ESCs to self renew in culture while retaining their developmental potential allows the production of unlimited numbers of differentiated cells to replenish those lost during disease27,28
. An alternative use of ESCs is to provide insights into disease mechanisms29,30
. ESCs carrying the genes responsible for a particular disease can be induced to differentiate into the cell types affected in that disease. Studies of these differentiated cells in culture could provide important information regarding the molecular and cellular nature of events leading to pathology.
We have used this approach to develop an in vitro
model of amyotrophic lateral sclerosis (ALS). ESC lines were derived from normal mice, and from mice that overexpress the wild-type human SOD1
transgene or the mutant SOD1G93A
transgene, the latter of which is responsible for one type of familial ALS. Using previously established methods12
, the ESC lines were differentiated into motor neurons in culture. The wild-type SOD1
and the mutant SOD1G93A
motor neurons produced high levels of the corresponding human SOD1 proteins, and they both showed properties of bona fide
motor neurons. These motor neurons could be maintained in long-term culture, providing the opportunity to detect differences between the mutant SOD1G93A
motor neurons and those derived from control cell lines.
A number of changes characteristic of neurodegeneration in ALS were observed in the mutant SOD1G93A motor neurons between 14 and 28 d. First, the intracellular localization of the SOD1G93A protein changed, forming inclusions that increased in size and density. Second, the amount of ubiquitin increased. Third, some motor neurons expressed activated caspase-3 and showed cytoplasmic staining with cytochrome c antibodies. Finally, a significant difference in survival was observed between mutant SOD1G93A motor neurons and the controls. Thus, many of the late-onset pathologies observed in both human ALS and SOD1G93A mice are recapitulated in this in vitro model, including the loss of motor neurons, which is the ultimate cause of symptoms in humans.
Several studies have suggested that cells in the spinal cord may have pathological, non–cell autonomous effects on motor neurons or on the rate of disease progression10,11
. However, these studies either were unable to identify the cell types that caused these effects10
or could not determine whether they acted directly to affect motor neuron survival11
. We found that cultures of ESC-derived motor neurons contain other cell types, including astroglia, and thus considered the possibility that these ESC-derived cells have a non–cell autonomous effect on motor neuron survival in vitro
. We therefore systematically examined the effects of coculturing motor neurons with primary glia from SOD1G93A
mice and from mice expressing the wild-type SOD1 protein. We found that mutant SOD1G93A
glia reduced the survival of both wild-type and mutant motor neurons. However, the effect was significantly greater on mutant SOD1G93A
motor neurons. Therefore, our studies show for the first time that an ALS genotype in glial cells directly and negatively affects the survival of motor neurons, and confirm that there is a non-cell autonomous component to motor neuron degeneration.
Consistent with the results reported here, another study described in this issue has shown that primary astrocyte cultures expressing ALS-associated mutant SOD1 proteins contain diffusible factor(s) that are toxic to primary and ESC-derived motor neurons31
. In this study, motor neurons were the only cell types affected by these mutant glial cells and only SOD1G93A
glial cells, not muscle cells or fibroblasts, adversely affected motor neuron survival. Although mutant primary neurons showed morphometric alterations, their survival up to 14 d in culture was indistinguishable from that of their wild-type counterparts. In our studies, differences in survival between wild-type SOD1
and mutant SOD1G93A
ESC-derived motor neurons were observed at 14 and 28 d in culture. The differences between the two studies may therefore originate in the source (embryo- or ESC-derived) or number of the motor neurons used, and the timeframe of the investigations.
The results reported here and elsewhere in this issue31
provide the basis for detailed mechanistic studies of the interactions between SOD1 mutant motor neurons and glia, and they provide an assay for diffusible factor(s) that are either toxic or beneficial for motor neuron survival. The model system described here may also provide a high-throughput cell-based assay for small molecules that promote survival of mutant SOD1 motor neurons. Finally, these studies validate the use of ESCs carrying disease-causing genes to define disease mechanisms. In this regard, our work suggests that the future development of human ESC lines from patients via somatic cell nuclear transplantation could provide the opportunity to study the nature of sporadic ALS, which affects the majority of ALS patients.