Northwestern University-led researchers have created the first highly mature neurons from human induced pluripotent stem cells (iPSCs). Although previous researchers have differentiated stem cells to become neurons, those neurons were functionally immature, resembling neurons from embryonic or early postnatal stages. By culturing iPSCs-derived neurons on coatings with “dancing molecules,” the Northwestern University team created neurons that demonstrated more functional maturity, enhanced synaptic signaling, electrical activity, and branching, and improved survival rates.

Such neurons could feasibly be transplanted into patients with spinal cord injuries or neurodegenerative diseases to replace lost or damaged neurons. And by advancing the age of human neurons, researchers might be able to better study adult-onset diseases in relatively simple and cost-effective cell cultures.

The scientists, headed by Northwestern professor Samuel I. Stupp, PhD, reported on their work in Cell Stem Cell, in a paper titled, “Artificial extracellular matrix scaffolds of mobile molecules enhance maturation of human stem cell-derived neurons.

The ability to generate iPSCs and promote their differentiation into neural cells has provided what the researchers termed unprecedented access to the human central nervous system (CNS). “It has enabled the assembly of models for the investigation of neurodevelopment and neurological diseases, which have led to significant advancements in our understanding of these processes,” they wrote. However, culturing stem cell-derived neurons in vitro remains challenging. Neurons grown in cell autonomous systems do not become fully mature, and they demonstrate reduced long-term viability. The limited maturation obtained with current stem cell culture techniques also diminishes their potential use in neurodegeneration studies.

“When you have an iPSC that you manage to turn into a neuron, it’s going to be a young neuron,” said Stupp, who is co-corresponding author of the study. “But, in order for it to be useful in a therapeutic sense, you need a mature neuron. Otherwise, it is like asking a baby to carry out a function that requires an adult human being. We have confirmed that neurons coated with our nanofibers achieve more maturity than other methods, and mature neurons are better able to establish the synaptic connections that are fundamental to neuronal function.” Stupp is the Board of Trustees professor of materials science and engineering, chemistry, medicine and biomedical engineering at Northwestern, where he is founding director of the Simpson Querrey Institute for BioNanotechnology (SQI) and its affiliated research center, the Center for Regenerative Nanomedicine. Stupp has appointments in the McCormick School of Engineering, Weinberg College of Arts and Sciences, and Feinberg School of Medicine.

To develop culture conditions that better mirror the microenvironment of the nervous system it’s important to consider the extracellular matrix (ECM), an “intercellular scaffold” that the authors stated plays “a pivotal role in neuronal maturation, signaling, and aging.” For their work to generate mature neurons from stem cells, the team created an “ECM-mimetic platform” based on the breakthrough “dancing molecules,” technology introduced last year by Stupp, which comprises scaffolds of supramolecular nanoscale fibrils formed by peptide amphiphile (PA) molecules. Stupp’s lab developed the material as a potential treatment for acute spinal cord injuries. In previous research, Stupp discovered how to tune the motion of molecules, so they can find and properly engage with constantly moving cellular receptors. By mimicking the motion of biological molecules, the synthetic materials can communicate with cells.

A key innovation of Stupp’s research was discovering how to control the collective motion of more than 100,000 molecules within the nanofibers. Because cellular receptors in the human body can move at swift rates—sometimes at timescales of milliseconds—they become difficult-to-hit moving targets. “Imagine dividing a second into 1,000 time periods,” Stupp said. “That’s how fast receptors could move. These timescales are so fast that they are difficult to grasp.”

For their newly reported work, the team first differentiated human iPSCs into motor and cortical neurons, and then placed them onto coatings of the synthetic nanofibers containing these rapidly moving dancing molecules. The investigators found that nanofibers tuned to contain molecules with the most motion led to the most enhanced neurons. In other words, neurons cultured on more dynamic coatings—essentially scaffolds composed of many nanofibers—were also the neurons that became the most mature. “We observed that supramolecular scaffolds with similar nanofiber architecture and chemical composition displayed remarkable enhancement of bioactivity when they exhibited more intense supramolecular motion,” the investigators reported. “Neurons cultured on matrixes with highly mobile PA molecules exhibited several features consistent with increased functional maturation.”

“The reason we think this works is because the receptors move very fast on the cell membrane and the signaling molecules of our scaffolds also move very fast,” Stupp said. “They are more likely to be synchronized. If two dancers are not in sync, then the pairing doesn’t work. The receptors become activated by the signals through very specific spatial encounters. It also is possible that our fast-moving molecules enhance receptor movement, which in turn helps cluster them to benefit signaling.”

The investigators found that not only were the resulting enriched neurons more mature, but they also demonstrate enhanced signaling capabilities, and greater branching ability, which is required for neurons to make synaptic contact with one another. The authors further noted, “Our results suggest an important role for motion in cell signaling beyond simple conformational changes of individual molecules.” And, unlike typical stem cell-derived neurons which tend to clump together, these neurons did not aggregate, making them less challenging to maintain. “The PA-based ECM-mimetic technology we describe here offers biological and technical advantages relative to current approaches for culturing stem-cell-derived neurons in vitro,” the team wrote. “Our work demonstrates the importance of incorporating dynamically controllable features into synthetic ECM scaffolds that can provide significant improvements to stem-cell-based neuronal models.”

With further development, the researchers believe the mature neurons could be transplanted into patients as a promising therapy for spinal cord injuries as well as neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, or multiple sclerosis.

“This is the first time we have been able to trigger advanced functional maturation of human iPSC-derived neurons by plating them on a synthetic matrix,” said Northwestern’s Evangelos Kiskinis, PhD, co-corresponding author of the study. “It’s important because there are many applications that require researchers to use purified populations of neurons. Most stem cell-based labs use mouse or rat neurons co-cultured with human stem cell-derived neurons. But that does not allow scientists to investigate what happens in human neurons because you end up working with a mixture of mouse and human cells.” Kiskinis is an assistant professor of neurology and neuroscience at Northwestern University Feinberg School of Medicine, a New York Stem Cell Foundation-Robertson Investigator, and a core faculty member of the Les Turner ALS Center.

Stupp and Kiskinis believe their mature neurons will give insights into aging-related illnesses and become better candidates for testing various drug therapies in cellular cultures. Using the dancing molecules, the researchers were able to advance human neurons to much older ages than previously possible, enabling scientists to study the onset of neurodegenerative diseases. “The composition of the ECM in the CNS is known to change with age … and could be mediating aspects of age-associated neurodegeneration,” the scientists noted. “Future efforts guided toward creating artificial coatings that mimic the aging ECM could prove useful.”

As part of the research, Kiskinis and his team took skin cells from a patient with ALS and converted them into patient-specific iPSCs. Then, they differentiated those stem cells into motor neurons, which is the cell type afflicted in this neurodegenerative disease. Finally, the researchers cultured neurons on the novel synthetic coating materials to further develop ALS signatures. Not only did this give Kiskinis a new window into ALS, these “ALS neurons” also could be used to test potential therapies.

“For the first time, we have been able to see adult-onset neurological protein aggregation in the stem cell-derived ALS patient motor neurons,” Kiskinis said. “This represents a breakthrough for us. It’s unclear how the aggregation triggers the disease. It’s what we are hoping to find out for the first time.”

“We anticipate that the PA platform will be of great interest to the stem cell community focused on developing hiPSC-based models of neurodevelopmental, neurological, and neurodegenerative diseases,” the investigators stated. “The adaptive nature and inherent flexibility in the design of the PA supramolecular materials can facilitate the development of additional ECM-mimetic platforms in the future.”

Further down the road, it may be possible for iPSC-derived mature, enhanced neurons to be transplanted into patients with spinal cord injuries or neurodegenerative diseases. For example, physicians could take skin cells from a patient with ALS or Parkinson’s disease, convert them into iPSCs and then culture those cells on the coating to create healthy, highly functional neurons.

Transplanting healthy neurons into a patient could replace damaged or lost neurons, potentially restoring lost cognition or sensations. 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 challenging for a disease like ALS, as transplanted motor neurons in the spinal cord will need to project their long axons to the appropriate muscle sites in the periphery but could be more straightforward for Parkinson’s disease,” Kiskinis commented. “Either way this technology will be transformative.”

Stupp added, “It is possible to take cells from a patient, transform them into stem cells and then differentiate them into different types of cells. But the yield for those cells tends to be low, and achieving proper maturation is a big issue. We could integrate our coating into large-scale manufacturing of patient-derived neurons for cell transplantation therapies without immune rejection.”


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