A team of researchers have developed a powerful lab model to better understand how muscles and neurons connect in the fight against Neuromuscular Diseases such as ALS.
Neuromuscular diseases are crippling and, for the most part, incurable, affecting 160 people out of every 100,000 worldwide. Disorders such as ALS and multiple sclerosis have an impact on muscle function, resulting in muscle waste and loss of motor function. The fact that it is notoriously difficult to grow tissue in a lab that shows the connection between our muscles and the neurons that control them is a major impediment in the fight against these diseases. That is, until now.
A USC Viterbi School of Engineering biomedical engineering Ph.D. student collaborated with USC Keck School of Medicine and USC Dornsife College of Letters, Arts, and Sciences researchers to develop a vastly improved new lab-grown tissue model that provides a more stable view of the neuromuscular junction — an important part of our system that translates electrical impulses generated by muscles.
The study was led by Jeffrey Santoso, a Ph.D. student in the Laboratory for Living Systems Engineering, which is led by Megan McCain, the Chonette Early Career Chair and associate professor of biomedical engineering, stem cell biology, and regenerative medicine.
The neuromuscular junction, according to Santoso, is a highly structured connection with pretzel-like folds that increase the area for communication between a neuron and a muscle.
“The neuromuscular junction is the space where the neuron will release signaling molecules called neurotransmitters, which will then bind onto receptors that are located on the surface of the muscle fibers,” Santoso said.
“And when these molecules attach to those receptors, it causes the muscle cell to be depolarized — so there’s a voltage change — and that’s what causes your muscles to contract.”
There is often stress or a break in connection within the neuromuscular junction in neuromuscular diseases, as well as in the natural ageing process. Accurate lab-grown tissue models are critical for understanding age-related degeneration, as well as the progression of neuromuscular disease and the most effective treatments. However, when trying to grow muscle fibers and neurons together in lab models, researchers have struggled to replicate this complex connection, or innervation point.
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“Traditionally, when muscle and neurons are grown together in a dish, the expected structure at the innervation point is absent,” Santoso said, “leading to a functionally inept tissue that does not represent proper physiology, making it hard to draw conclusions about diseases and potential drugs.”
As a result, previous tissue models usually die within two weeks. Santoso and his co-authors developed a gelatin-based gel platform that allows skeletal muscle to grow in the lab in its proper aligned architecture, maintaining its structure for at least a month, and generating contractions comparable to the strength of native human tissue.
“When you try to grow the tissue in the lab, it usually involves growing muscle cells in a layer and putting the neuron cells on top,” Santoso said.
“The junction that forms doesn’t create a structure of these pretzel-like folds, and so this causes a variety of issues, because it might not respond to certain drugs as you would expect.”
Santoso and his colleagues combined lab-grown skeletal muscle with Ichida lab-developed human stem cell-derived motor neurons. They discovered that the muscle was successful in forming more structured neuromuscular junctions, which were characterized using the Dickman lab.
Previous tissue models, according to Santoso, frequently used stiff plastic or glass dishes that did not provide the right kind of protein environment for the tissue and frequently caused muscle cells to peel away when contracted, resulting in cell death.
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“The gelatin hydrogel that we’re using, as a natural biomaterial, is naturally adhesive to the skeletal muscle cells, and this is one reason we were able to see our tissue model maturing much further than a lot of other previous models,” Santoso said. “The skeletal muscle can survive on the surface for a longer period of time, and having this increased time in the culture means that when we introduce the motor neurons to the culture it can fully integrate to create that neuromuscular junction.”
Because the human neuromuscular system is so complex, Santoso said that studying neuromuscular disease was especially difficult. Motor neurons are found in our spinal cords and must travel through a complex network of connections to reach all of our muscles.
“There are so many places in this pathway where things can go wrong,” Santoso said. “Because of this, you need that laboratory model to be pretty close in function and structure, compared to what you see in living beings.”
This project was originally funded by an Eli and Edythe Broad Innovation Award in Stem Cell Biology and Regenerative Medicine at USC to the McCain, Ichida, and Dickman labs.
The multidisciplinary research team is now looking to broaden their collaboration so that they can use their engineered tissue platform to model a variety of human diseases using patient-derived cells.
“At the moment, neuromuscular diseases only really have palliative treatments, where they can only delay the severity of some of the symptoms. There’s not really a good way to reverse or prevent the damage.” Santoso said.
“Making these kinds of models and trying to parse out the mechanisms of the disease might give us a better idea of how we can develop therapeutics to better alleviate the symptoms, or ideally, to remove them.”
Image Credit: Image/USC Laboratory for Living Systems Engineering
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