IN VITRO MODEL SYSTEMS FOR INVESTIGATION OF MOTONEURON DEVELOPMENT AND DISEASE

Abstract

Spinal motoneurons innervate muscle fibres and represent the final component in the motor pathway responsible for producing movement by eliciting muscle contraction. Although the anatomy and physiology of motoneurons have been well studied, many questions remain about how these cells develop or why (and how) they die in motoneuron diseases such as amyotrophic lateral sclerosis (ALS). In this thesis, I present data that supports the use of several in vitro approaches to investigate these issues. The first approach uses motoneurons derived from mouse induced pluripotent stem (iPS) cells – stem cells that can be generated from differentiated cells. Using a wide range of experimental approaches I show that motoneurons derived from iPS cells develop the same cellular, anatomical, behavioural and electrophysiological properties as their endogenous counterparts. iPS cell-derived motoneurons fire repetitive action potentials with the same firing pattern as endogenous motoneurons, extend axons out of the neural tube when transplanted in ovo and make functional connections with muscle fibres in vitro and in vivo. In a second approach, I show that motoneurons derived directly from fibroblasts without a stem cell intermediary step (termed induced motoneurons) develop several characteristics typical of normal motoneurons. These include the ability to extend axons out of the developing spinal cord when transplanted in ovo and make connections with muscle fibres in vitro. Finally, I established a differentiation protocol to preferentially generate a very specific subtype of motoneurons that selectively target dorsal limb muscles during development. Like their endogenous counterparts, these embryonic stem cell derived motoneurons (ESCMNs) express Lhx1 and FoxP1, develop large soma sizes and form large neuromuscular junctions. As occurs in patients with ALS, I show results that suggest that large ESCMNs expressing the G93A SOD1 mutation are more susceptible to death than smaller ESCMNs when co-cultured with muscle fibres. In summary, in this thesis I have developed, and critically examined, several in vitro models useful for studying motoneuron development and motoneuron diseases. While additional studies are warranted, I believe that these model systems provide novel ways to study ALS and to possibly become a source of motoneurons for cell replacement therapies.