Scientists have made strides turning stem cells into motor neurons, but a motor neuron by itself is not much use—it’s only when it innervates a muscle that it can get things moving. Now, for the first time, researchers claim that stem cell-derived motor neurons make electrically active connections with co-cultured muscle cells. Researchers who reported the new model system in the May 4 PLoS ONE are interested in using it to study spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), both of which involve impairment at the neuromuscular junction, said senior author Bennett Novitch, University of California, Los Angeles.
With the advent of stem cell and induced pluripotent stem cell (iPSC) technology, scientists are studying many neurological diseases in culture (see ARF related news story). However, researchers have mainly examined neurons in isolation or in co-culture with glia, Novitch said. While stem cell-derived motor neurons can innervate the muscles of mice and chick embryos (Yohn et al., 2008; Peljto et al., 2010), it has been challenging to evaluate the electrical function of these new, stem cell-based junctions. The tightly packed tissue makes it hard to isolate a single neuron-muscle pair to analyze. Novitch teamed with faculty colleague and first author Joy Umbach to create neuromuscular junctions in vitro so they could measure junction function.
The researchers tested many different parameters to find those that best nurtured cell survival and synapse formation. For Umbach and Novitch’s goal of studying the electrophysiology of the synapse, the most crucial part of the protocol was to plate the neurons and muscle cells sparsely. Sparse turns out to be approximately 24,000 cells per 35-millimeter dish. Higher densities made it difficult to patch-clamp an isolated pair of cells and obtain clear recordings, Novitch said.
Umbach and colleagues found that where the motor neurons and muscles met, they made synapses that included many of the proteins found in natural neuromuscular junctions, such as acetylcholine receptors and synaptic vesicle proteins. Stimulating the neurons created an action potential and, a few milliseconds later, an excitatory current in the associated muscle cells. Examining the kinetics, “they seem to be remarkably similar to what one gets with [natural] tissues,” Novitch said, such as isolated nerve-muscle preps (Fatt and Katz, 1951).
The researchers next plan to test some diseased cell types in their system, such as iPS-based neurons from people with ALS or SMA. Novitch hopes the culture could be used to screen for medicines that would block denervation, the process that robs people with ALS of their ability to control their skeletal muscle. The neuromuscular junction has not been closely examined in studies of motor neuron disease, and may turn out to be an important site of action, commented Arnold Kriegstein of the University of California, San Francisco, who was not involved with the study.
However, Kriegstein suggested that the current in-vitro model is not very robust, because the junctions only lasted for a week. This is the most important limitation of the artificial system, Novitch agreed; natural synapses, of course, last much longer. Novitch suspects that adding more elements of the neural circuit—such as Schwann cells, glia, and interneurons—will help stabilize the connections. The team has also found, in not-yet-published experiments, that neuromuscular junctions derived from human embryonic stem or iPS cells last for several weeks, suggesting the mouse synapses, in particular, are unstable.
Umbach JA, Adams KL, Gundersen CB, Novitch BG. Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons. PLoS ONE. 2012 4 May. 7(5):e36049. Abstract
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