Over the last two years, researchers at the Institute for Stem Cell and Regenerative Medicine (ISCRM) have been using skeletal muscle derived from induced pluripotent stem cells (iPSC) to study how neuromuscular diseases develop and to test potential therapeutics. However, there is an important shortcoming.
Current approaches do not provide a full picture of neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, and myasthenia gravis because it does not recapitulate the synapses that connect the nervous system to the musculature: the so-called “neuromuscular junction.” It is the deterioration of these junctions that causes patients with these diseases to lose the ability to walk, talk, and eventually breath.
“Growing neurons or muscle in a dish works well if you want to study a purely neuronal or purely muscle disease,” says Alec Smith, PhD, an ISCRM faculty member and Research Assistant Professor in the Department of Physiology and Biophysics. “But if you want to effectively model diseases that cause a breakdown of the neuromuscular junction, you need a tool that allows those two cell types to interact in culture.”
With that in mind, Smith has been partnering with the Mack and Bothwell Labs, part of the Neuromuscular Disease Research Group, to engineer a system that would enable them to explore that connectivity in a way that closely models neuromuscular junction function in the body and that is conducive to more high-throughput screening than current laboratory methods.
Testing Potential Therapies for Neuromuscular Diseases
Now, Smith has received two sources of funding to support this effort. First, with a UW Royalty Research Fund grant, the ISCRM researchers will develop the NMJ modeling technology in partnership with Seattle biotech Curi Bio. Second, an NIH grant will allow the team to use the technology, along with cell lines developed by Claire Clelland, MD, PhD at the University of California San Francisco, to test potential therapeutics for an inheritable form of ALS.
The system that will enable more detailed study of neuromuscular junctions is based on technology developed by ISCRM Associate Director Nate Sniadecki, PhD, a Professor of Mechanical Engineering. In that system, which was created to measure the force produced by engineered cardiac tissues, cell-laden scaffolds are arrayed between two posts – one rigid, one flexible. When the cells within the engineered tissues contract, the force is measured by magnetic sensors.
Smith and his collaborators have now adapted this technology to measure responses in engineered skeletal muscle tissues and hope to soon finalize further modifications that will facilitate measurement of nerve-muscle interactions.
Once it is finalized, this highly automated technology will enable researchers to monitor dozens of engineered muscle tissues simultaneously, which makes it possible to test many different promising drugs at once. Smith and his collaborators can set up the software to record every 30 minutes for ten hours, for example, a significant improvement over previous, more laborious, approaches. The ability to precisely monitor engineered muscle activation in response to neuron stimulation will allow the investigators to rapidly and accurately assess the health of the synaptic connections that form between neurons and skeletal muscle cells.
Smith explains that Curi Bio is building the physical infrastructure that will house the cells while the ISCRM researchers are responsible for growing the muscle cells and neurons, determining how to optimally stimulate them to interact, and validating the magnetic two-post technology as a platform in which to study neuromuscular disease pathology.
Moving Therapies to the Clinic
The team has already published evidence that Sniadecki’s technology is useful for muscle researchers. In a paper in the Journal of Tissue Engineering, the researchers showed that the magnetic sensing system could be adapted to analyze engineered skeletal muscle tissues derived from both iPSC and primary sources and to measure the performance of muscle over time as tissues in the lab are exposed to a variety of drugs.
Specifically, the researchers reported on changes in contraction magnitude dependent on the frequency of electrical stimulation – a characteristic present in native muscle tissue and critical to demonstrating that engineered tissues mirror the function of the real thing. “The findings of the paper lead us right into the next step, which is integrating two cell types – neurons and skeletal muscle,” says Smith.
The process of integrating neurons into the experiment will be funded by an R03 grant from the NIH, Smith’s first as an independent investigator. The R03 grant builds on support Smith received as a KL2 scholar through a grant administered by the Institute for Translational Health Sciences. That program provides the time, funding, mentorship, and training necessary to foster the early career development of clinical and translational researchers.
“The R03 is about using the magnetic sensing technology to evaluate the neuromuscular interactions that go awry in diseases we are studying,” he says. “We’ll take the gene-edited cell lines from our UCSF partners and run them through our system. The goal is to identify which gene therapy strategy is most effective at improving the function of the diseased neuromuscular junction.”
Looking ahead, Smith has a vision for continued progress. “By the end of the grant period, we would like to be at a stage where we have built and validated a tool that integrates skeletal muscle cells and neurons and have used that platform to show that one or more of the gene therapy models from UCSF has therapeutic potential. If it does, it will increase the speed with which we can move promising therapies toward the clinic in the future.”