Hanging up their dancing shoes was not an option, so in 2003, patients with Lou Gehrig’s disease joined a clinical trial to see if a muscle-strengthening supplement called creatine monohydrate could slow the deterioration of their mobility. The supplement had shown promising results in mice, but unfortunately the drug impacted neither the patients’ rate of decline nor their survival. It was a disappointment for the participants as well as for the scientists whose work had fallen flat.
Animals are a vital part of drug discovery, but they are not perfect surrogates for humans. Furthermore, research with mice is expensive and raises ethical and moral dilemmas. For these reasons, scientists at Los Alamos National Laboratory, along with external collaborators, have spent more than a decade developing multiple lab-based, in vitro organ platforms to reduce our reliance on animal research. Their most recent endeavor is to recreate a neuromuscular junction (NMJ): the critical intersection necessary for all muscle movements, from voluntary leaps and pirouettes to involuntary heartbeats.
Neuromuscular junctions are critical for movement—from voluntary leaps and pirouettes to involuntary heartbeats.
Neuromuscular junctions are where motor neuron cells communicate with muscle cells, but their interaction is complex and difficult to replicate. Robust, in vitro NMJ platforms could change everything. They could enable a thorough examination of NMJ function, but more than that, they could also facilitate the rapid screening of thousands of potential drugs for Lou Gehrig’s disease and much, much more. Simple, inexpensive ways of screening therapeutics could ultimately improve or save millions of lives.
Motion = life
In a normally functioning NMJ, an electrical impulse in a motor neuron and an influx of calcium trigger the release of a chemical messenger called acetylcholine (ACH). ACH molecules enter the junction between the neuron and muscle and bind to external receptors on the muscle cell, initiating a cascade of cellular events that cause the muscle fibers to contract. A regulatory enzyme called acetylcholinesterase (AChE) can also be released to break down the ACH when necessary, allowing the muscles to relax. There are over 600 muscles in the human body—skeletal, cardiac, and smooth—and this careful balance of releasing and eliminating molecules enables each one of them to move.
Malfunction at the junctions, however, is a key aspect of many disorders, including Lou Gehrig’s disease, which is scientifically known as Amyotrophic Lateral Sclerosis (ALS). In ALS patients, neurons break down over time and lose their ability to send ACH to muscles. Without this stimulation, the muscles atrophy and deteriorate, decreasing mobility and ultimately affecting a patient’s ability to waltz, walk, pump blood, and even breathe. Scientists have studied ALS for more than 100 years, but there are still very few treatments available, and there is no cure; ALS is always fatal.
On the other hand, an overabundance of muscle stimulation is also a problem. Nerve agents and certain pesticides impact NMJ communication by inhibiting AChE, so the ACH messengers are never removed from the junction and the muscles contract uncontrollably. Victims of nerve agent exposure experience an overstimulation of every NMJ system, causing vomiting, cramping, salivation, hypertension, or convulsions—potentially leading to asphyxiation or cardiac arrest.
“We want to improve countermeasures for these poisonings,” says Los Alamos biologist Jennifer Harris. “And we want to develop an ethical platform to use for testing.”
With this in mind, Harris and fellow Los Alamos biologist Rashi Iyer, working with the Defense Threat Reduction Agency (DTRA), convened a team in 2016 to build an in vitro NMJ to study potential countermeasures for nerve agent poisonings, therapeutics for ALS, and more. Since then, their work has expanded into multiple projects and their advances have led to a thorough understanding of the conditions and genes needed to grow both mouse and human NMJs. Furthermore, it has led to a high-throughput screening platform that is poised to significantly affect neuromuscular drug discovery.
Seeking signals
Growing cell cultures in a laboratory environment is complicated because cells within living organisms rely on signals from each other and from their environment. Most cells in the human body carry an entire set of the genetic information needed to create the organism (the exception being reproductive cells such as eggs and sperm that only carry half the information). With all the genetic data present during development, embryonic stem cells can differentiate into anything—lung cells or heart cells or skin cells—based on which genes are activated during which stages of maturation. Cells communicate with each other by sending small molecules, such as proteins or sugars, as signals to initiate this gene activation.
Animals are a vital part of drug discovery, but they are not perfect surrogates for humans.
In addition to signals from other cells, environmental cues such as changes in temperature, pressure, or pH can prompt a cell to repair damage, initiate enzyme activity, and so forth. Cell signaling is important beyond the development process as mature cells also rely on chemical messages. Together, these requirements make it very difficult to remove cells from a living organism and grow them in a petri dish or other ex-vivo laboratory environment. Without the correct mixture of signals, the cells can die.
For these reasons, scientists generally use stem cells or other cells that are specifically adapted for growing in culture. With an NMJ, however, the Lost Alamos team faced the challenge of combining two completely different cell cultures—muscle cells and neurons—that require two different sets of signals to create a mature, functioning NMJ.
Starting with immature mouse cell lines, which grow rapidly and are readily available and well studied, Harris and Iyer’s team researched the types of small molecules known to help neurons and muscle cells mature. Then, mimicking the natural process of cell communication, the scientists added a mix of these molecules (known as a culture medium) hoping the immature cells would further mature and form an NMJ. But the process wasn’t entirely straightforward. For instance, adding muscle cells to a neuron culture did not work at all.
Fluorescent staining confirms the formation of neuromuscular junctions in mouse cells. Here, the Los Alamos team observed the presence of acetylcholine molecules (pink), indicating the muscle cells (green) and neurons (red) were beginning to interact by establishing neuromuscular junctions. Staining of cell nuclei (blue) confirms that muscle and neuron cells are mature.
“We knew that muscle cells need glucose and appropriate levels of serum,” says Los Alamos biologist Sofiya Micheva-Viteva. (Serum is a biochemical mixture of growth factors and proteins.) “But the serum made the neurons divide more, instead of completing their differentiation and maturing.”
“Thus began a lot of trial-and-error work to determine how to co-culture neurons and muscles,” says Iyer. This involved iterating to find the best culture medium for each type of cell, as well as the timing of how long the cells should grow separately before being put together to mature into an NMJ. The painstaking work paid off, and the Los Alamos team identified an optimal seven-day process to create a functioning mouse NMJ.
To grow a human NMJ, the team used a similar media-based approach to mature induced pluripotent stem cells (IPSCs) into neurons and muscle cells. IPSCs are derived from adult human cells, but are similar to embryonic stem cells in that they are not differentiated and—based on the signals they receive—can be coerced into becoming whatever type of cell is needed for a study.
It took a lot of trial-and-error work to determine how to co-culture neurons and muscles.
“We discovered that it is an amazingly complex process to form a human NMJ,” says Harris. “So a big part of our project has been to elucidate the signals that are responsible for development.”
Elucidating the signals meant taking a deeper look into the specific genes being activated by the small-molecule messages. When a developing muscle cell receives a signal from its neighbors to begin growth, genes in its nuclear DNA “turn on” by sending RNA transcripts out into the cell to make enzymes, regulatory proteins, etc. Studying these the transcripts—a field called transcriptomics—can reveal which genes are active at a specific point in development. By doing transcriptomic analysis of their cultures, the Lab team sought to connect the dots to understand which small-molecule signals might be responsible for turning on which genes.
This image of human neuromuscular junctions shows muscle cells (green), neurons (red), and the nuclei (blue) for both cell types. The two cell types were grown together for the final steps of differentiation, which took approximately 16 days.
The human NMJ worked, but it took over a month to mature using culture media. The scientists could visually observe the muscle cells and neurons growing close together and could confirm the presence of some ACH molecules through staining, which meant the cells were demonstrating some functionality. However, the team continued to research ways to do better.
“If we could just turn on the right genes at the right time to make an NMJ quickly, then we could test and study lots of things,” says Harris.
Genetic scissors
In 2020, two scientists received the Nobel Prize in Chemistry for their pioneering work on a technology known as CRISPR-Cas9. This technology—metaphorically referred to as genetic scissors—uses an enzyme to cut and edit genes to activate certain functions within organisms. Since about 2014, scientists have been exploring the use of CRISPR systems in a wide range of research endeavors from agriculture to gene therapy.
“So, we asked ourselves,” says Iyer, “can we use CRISPR to turn on a gene instead of a signaling molecule?” Through Laboratory-directed funding, Iyer and Micheva-Viteva joined Lab biologist Scott Twary and others in 2018 to investigate using CRISPR, instead of culture media, to initiate the maturation of IPSCs.
The benefits of CRISPR are many. Twary explains that when using culture media to mature cells, there could be variation in their responsiveness. Using CRISPR, all cells of a particular type would receive the same instructions to mature, thus ensuring a uniform response and a more homogenous population. Hopefully, it would also speed up the process in a reliable way.
“To differentiate motor neurons, I know it will take 28 days, four types of culture medium, and seven different small molecules to activate and inhibit specific pathways,” explains research technologist Emilia Solomon. Expecting CRISPR could streamline the process, the team began to systematically compare the two growth strategies: media versus CRISPR. Using known regulatory proteins as targets for CRIPSR gene editing, the scientists prompted the motor neurons and muscle cells to mature separately and studied them at each step of development using transcriptomics to determine which genes were actually active.
One regulatory protein in particular, called MYOD1, is known to be key to muscle cell development. Using CRISPR, the Los Alamos scientists inserted a modified version of MYOD1 that could be controlled by an additional chemical.
“Normally a whole series of genes is activated to mature a muscle cell, but we can skip some of the steps by activating MYOD1,” says Laboratory postdoctoral researcher Joseph Sanchez. Sanchez explains that controlled activation of MYOD1 caused the cells to differentiate into muscle in half the time: the cells showed signs of maturation at day 12 that were similar to those at day 28 for media-activated cells. The CRISPR approach, however, was not as advantageous with neurons, and the team is still evaluating its overall effectiveness.
“The neurons did not mature as fast and they didn’t last,” explains biologist Katie Davis-Anderson. “We were able to speed up the process to detect some neuron function but the activity ended quickly. We are still learning how to modify these cells but also recognizing that forced maturity might not last.”
Part of this continued learning has been to co-culture the neurons and muscle cells for longer periods of time (after first differentiating separately) to capitalize on their natural cell signaling. However, Twary warns that it is not yet clear if there are consequences to skipping some of the developmental steps. He explains that an important part of the CRISPR project is to identify and clarify if there are any secondary effects to using CRISPR that the scientific community should be aware of.
Rapid results
Although the use of CRISPR to facilitate NMJ growth is still being considered, other members of the NMJ team have made significant progress with a separate project to use their optimized, media-matured mouse cells to develop a drug screening platform. The platform would allow scientists to quickly determine whether a potential drug is working by directly detecting NMJ function in lab-grown cells instead of using live mice.
When muscle cells mature, they can sometimes spontaneously twitch. When cultured with motor neurons, the scientists must be able to distinguish between those spontaneous twitches and any movements initiated by a neuron through an NMJ. In some in vitro models, scientists have used a so-called “patch clamp” to attach wires to a neuron to measure its electrical potential, thus confirming the neuron as the cause of muscle twitching; however, the Los Alamos team wanted something better.
“The patch clamp is invasive. It only measures one cell at a time, it’s time consuming, and its use requires lots of training,” says Solomon. “And it destroys the cell.” To avoid the patch clamp method, Solomon explains that the Los Alamos team developed a unique approach using genetic modification and a commercially available multi-electrode array (MEA) system.
(Left) The Los Alamos team developed a unique approach to quickly measure if a potential drug is working by directly detecting neuromuscular function in lab-grown mouse cells using genetic modification and a commercially available multi-electrode array (MEA) system. The neuromuscular junction cells are modified to respond to light and then grown in the wells of the MEA culture dish so their movement can be detected. (Right) Motor neurons are growing in the wells on top of electrodes (black lines) for detection.
Building on their successful method of growing functional mouse NMJs, the Los Alamos scientists genetically modified both types of cells to enhance their use in a screening platform. First, they modified the neurons with a light-activated protein so that the neurons could be stimulated on command. Second, they modified the skeletal muscle cells with a heart protein that would help amplify the electrical signal once the muscle is activated by the neuron. Finally, they grew the NMJs in the wells of an MEA system so that the electrodes underneath the cells could record the twitching of the entire muscle population.
With this system, scientists could study potential drugs by measuring the strength of the MEA signal. For instance, the scientists could artificially compromise the NMJ cells and then measure the effectiveness of various drugs by looking for an increased electrical signal (the NMJ regained function) or a diminished signal (the drug didn’t work). The system also has the potential to work using cells from an actual patient.
We could quickly screen hundreds to thousands of compounds against a poison or disease.
“This is one of our biggest achievements towards a high-throughput screening tool,” says Micheva-Viteva. “We can start with stem cells, grow them in one well using the same media, and use the multi-electrode array to measure NMJ activity.” Furthermore, if all of this can be done in just one well, then by multiplexing in a 96-well plate, the system lays the foundation for high-throughput screening of many potential drugs at once.
“Using our quickly growing mouse neuromuscular junctions, we could screen hundreds to thousands of compounds against a poison or disease and narrow down the best candidates,” explains Harris. “Then you can take the best ones and try them in the more complex human NMJ system.”
The Los Alamos NMJ team is currently optimizing this platform for newly funded toxicity studies as well, including one to detect botulinum toxin in food, which normally involves sacrificing hundreds of mice.
A full dance card
The high-throughput screening platform has the potential to profoundly improve drug development by speeding up the testing process. However, the Los Alamos NMJ team is also working to develop additional physiologically relevant models that could be useful for basic research.
Solomon explains that cultured mouse NMJs don’t last very long: generally, only a few weeks. After a while, the twitching muscle cells begin to pull away from the surface where they are growing. So, while a 2D NMJ might be revolutionary for rapidly screening thousands of drugs in a short time-frame, it does not precisely mimic the 3D conditions within a living organism for in-depth study. With this in mind, the team members are also creating a 3D model in which neurons and muscle cells grow in separate, spherical gel-like chambers with channels that allow the cells to communicate and eventually meet to form junctions.
This suite of reliable protocols for growing neurons, muscles, and even 2D or 3D NMJs has wide-reaching benefits beyond Los Alamos. For instance, understanding the unique conditions required for neuron growth could help scientists advance regenerative medicine to repair damaged or diseased tissues. Also, laboratories that specialize in personalized medicine could employ these growth protocols to culture a patient’s own cells for further study. Finally, new poisons or diseases could be quickly evaluated using improved cell-culture strategies. The advances made at the Lab set the stage for all of these possibilities.
“We want a model that is at the ready for any emerging need related to neuromuscular junctions,” says Iyer. And, by the look of things, that’s exactly what they’ve got.