Fluorescent images of human neurons (stained red, green and blue) growing on coatings with fast-moving molecules (left) or conventional laminin (right) for 60 days. The neurons spread homogeneously and showed more complex branching on the highly mobile coating developed at Northwestern. Credit: Northwestern University
Researchers have pushed the age limit of human neurons further than previously possible.
A team of researchers led by Northwestern University has achieved a breakthrough by producing the most mature neurons to date from human induced pluripotent stem cells (iPSCs). This breakthrough opens new avenues for medical research and the possibility of transplant therapies for conditions such as neurodegenerative diseases and traumatic injuries.
Previous efforts to turn stem cells into neurons have resulted in functionally immature neurons that resemble those in early stages of development. The limited maturation achieved with current stem cell culture methods limits their potential for studying neurodegeneration.
The study has just been published in the journal Cell Stem Cell.
To create the mature neurons, the team used “dancing molecules,” a groundbreaking technique introduced last year by Northwestern professor Samuel I. Stupp. The team first differentiated the human iPSCs into motor and cortical neurons, then placed them on synthetic nanofiber coatings containing the fast-moving dancing molecules.
Fluorescent image of a human neuron (red) growing on the coating with fast molecules (green) for 60 days. Credit: Northwestern University
Not only were the enriched neurons more mature, but they also demonstrated enhanced signaling abilities and greater branching ability, which is necessary for neurons to make synaptic contact with each other. And, unlike typical stem cell-derived neurons which tend to clump together, these neurons did not clump together, making them less difficult to maintain.
With further development, researchers believe these mature neurons could be transplanted into patients as a promising therapy for spinal cord injury as well as neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinsons, 
Fluorescent images of human neurons (stained red, green and blue) growing on coatings with fast-moving molecules (left) or conventional laminin (right) for 72 hours. Neurons attached and distributed evenly on the highly mobile coating but remained clumped together on the laminin coating. Credit:
Northwestern University
“This is the first time that we have been able to trigger advanced functional maturation of iPSC-derived human neurons by plating them on a synthetic matrix,” said Evangelos Kiskinis of Northwestern, co-corresponding author of the study. “This is important because there are many applications that require researchers to use purified neuron populations. Most stem cell-based labs use mouse or rat neurons co-cultured with neurons derived from human stem cells. But that doesn’t allow scientists to study what’s going on in human neurons, because you end up working with a mixture of mouse and human cells.
“When you have an iPSC that you manage to turn into a neuron, it will be a young neuron,” said Stupp, corresponding co-author of the study. “But, for it to be useful in a therapeutic sense, you need a mature neuron. Otherwise, it’s like asking a baby to perform a function that requires an adult human being. We have confirmed that neurons coated with our nanofibers mature more than other methods, and that mature neurons are better able to make the synaptic connections that are fundamental for neuronal function.
Kiskinis is assistant professor of neurology and neuroscience at
A key innovation of Stupp’s research was discovering how to control the collective movement of more than 100,000 molecules in nanofibers. Because cellular receptors in the human body can move at rapid speeds – sometimes on timescales of milliseconds – they become difficult moving targets to hit.
“Imagine dividing a second into 1,000 periods of time,” Stupp said. “It’s the speed at which the receivers could move. These time scales are so fast that they are difficult to grasp.
In the new study, Stupp and Kiskinis found that nanofibers tuned to contain the most mobile molecules led to the most enhanced neurons. In other words, neurons grown on more dynamic coatings—essentially scaffolds composed of many nanofibers—were also the most mature neurons, least likely to aggregate, and had more intense signaling abilities.
“The reason we think it works is because the receptors move very quickly across the cell membrane, and the signaling molecules in our scaffolds also move very quickly,” Stupp said. “They are more likely to be in sync. If two dancers are out of sync, pairing does not work. Receivers are activated by signals through very specific spatial encounters. It’s also possible that our fast-moving molecules enhance the movement of receptors, which in turn helps group them together to benefit signaling.
Neurons carrying the ALS signature open a new window on the disease
Stupp and Kiskinis believe their mature neurons will provide insight into aging-related diseases and become better candidates for testing various drug therapies in cell cultures. Using the dancing molecules, the researchers were able to advance human neurons to much older ages than before, allowing scientists to study the onset of neurodegenerative diseases.
As part of the research, Kiskinis and his team took skin cells from an ALS patient and converted them into patient-specific iPSCs. Then they differentiated these stem cells into motor neurons, which is the type of cell affected by this neurodegenerative disease. Finally, the researchers grew neurons on the new synthetic coating materials to further develop the ALS signatures. Not only did this give Kiskinis a new window into ALS, but these “ALS neurons” could also be used to test potential therapies.
“For the first time, we were able to observe an aggregation of neurological proteins in adulthood in motor neurons of stem cell-derived ALS patients. This represents a breakthrough for us,” Kiskinis said. “It is not known how the aggregation triggers the disease. This is what we hope to discover for the first time.
Hopes for future treatment of spinal cord injury, neurodegenerative diseases
Later, mature and enhanced iPSC-derived neurons could also be transplanted into patients with spinal cord injuries or neurodegenerative diseases. For example, doctors could take skin cells from a patient with ALS or Parkinson’s disease, convert them into iPSCs, and then grow those cells on the coating to create healthy, highly functioning neurons.
Transplanting healthy neurons into a patient could replace damaged or lost neurons, potentially restoring lost cognition or sensation. And, because the initial cells came from the patient, the new iPSC-derived neurons would genetically match the patient, eliminating the possibility of rejection.
“Cell replacement therapy can be very difficult for a disease like ALS, as motor neurons transplanted into the spinal cord will need to project their long axons to the appropriate muscle sites in the periphery, but it might be simpler for Parkinson’s disease. “, said Kiskinis. “Either way, this technology will be transformative.”
“It’s possible to take cells from a patient, turn them into stem cells, and then differentiate them into different cell types,” Stupp said. “But the yield of these cells tends to be low, and achieving adequate maturation is a big problem. We could integrate our coating into the large-scale fabrication of patient-derived neurons for cell transplantation therapies without immune rejection.
References: “Artificial extracellular matrix scaffolds of mobile molecules enhance maturation of human stem cell-derived neurons” by Zaida Alvarez, J. Alberto Ortega, Kohei Sato, Ivan R. Sasselli, Alexandra N. Kolberg-Edelbrock, Ruomeng Qiu, Kelly A. Marshall, Thao Phuong Nguyen, Cara S. Smith, Katharina A. Quinlan, Vasileios Papakis, Zois Syrgiannis, Nicholas A. Sather, Chiara Musumeci, Elisabeth Engel, Samuel I. Stupp and Evangelos Kiskinis, January 12, 2023, Cell Stem Cell.
DOI: 10.1016/j.stem.2022.12.010
“Bioactive scaffolds with enhanced supramolecular motion promote recovery after spinal cord injury” by Z. Álvarez, AN Kolberg-Edelbrock, IR Sasselli, JA Ortega, R. Qiu, Z. Syrgiannis, PA Mirau, F. Chen , SM Chin, S. Weigand, E. Kiskinis and SI Stupp, November 11, 2021, Science.
DOI: 10.1126/science.abh3602
The study was funded by the National Institutes of Health, the Les Turner ALS Foundation, the New York Stem Cell Foundation, the US Department of Energy and the Paralyzed Veterans of America Research Foundation.