Cell Stem Cell: Using iPSCs to create the first highly mature neurons

• By growing human-induced pluripotent stem cell-derived neurons on a coating with "dancing molecules," the researchers created mature neurons

• These neurons showed greater functional maturity, enhanced synaptic signaling, electrical activity, and branching, improved survival, and did not clump together

• These neurons can be transplanted into people with spinal cord injury or neurodegenerative diseases to replace lost or damaged neurons

• By raising the age of human neurons, researchers can study adult-onset diseases in relatively simple and cost-effective cell cultures

Northwestern-led researchers created the first highly mature neurons from human induced pluripotent stem cells (iPSCs), a feat that opens up new opportunities
for medical research and potential transplant treatments for neurodegenerative diseases and traumatic injuries.

While previous researchers have differentiated stem cells into neurons, these are functionally immature neurons, similar to embryonic or early postnatal neurons
.
The limited maturity gained by current stem cell culture techniques reduces their potential
for neurodegenerative disease research.

The study will be published Jan.
12 in the journal Cell Stem Cell.

To create full-fledged neurons, the team used "dancing molecules," a breakthrough technique
introduced last year by Northwestern University professor Samuel I.
Stupp.
The team first differentiated human iPSCs into motor neurons and cortical neurons, then placed them on a
synthetic nanofiber coating containing fast-moving dance molecules.

Abundant neurons are not only more mature, but also exhibit enhanced signaling and stronger branching abilities, which are necessary
for synaptic contacts between neurons with each other.
And, unlike typical stem cell-derived neurons that tend to clump together, these neurons don't cluster, making them less challenging
.

The researchers believe that with further development, these mature neurons could be transplanted into patients as a promising treatment
for spinal cord injuries as well as neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease or multiple sclerosis.

Mature neurons also provide new opportunities to study neurodegenerative diseases such as ALS and other age-related models
.
By increasing the age of neurons in cell culture, researchers can improve experiments to better understand late-onset disease
.

Evangelos Kiskinis, co-corresponding author of the study and Northwestern University, said: "This is the first time we have been able to trigger the advanced functional maturation
of human ipsc-derived neurons by plating them on a synthetic matrix.
" "This is important because there are many applications that require researchers to use purified neuronal populations
.
Most stem cell-based laboratories use mouse or rat neurons to co-culture with human stem cell-derived
neurons.
But that doesn't allow scientists to study what's going on in human neurons, because what you're ultimately looking at is a mixture
of mouse and human cells.
”

"When you have an iPSC and you manage to turn it into a neuron, it's going to be a young neuron, but, in order 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 to do.
We have shown that neurons wrapped in nanofibers are more mature than other methods, and mature neurons are better able to establish synaptic connections
that are critical to neuronal function.
”

Synchronized "dance" ability

To grow mature neurons, the researchers used special nanofibers, a potential treatment
for acute spinal cord injury developed in Stupp's lab.
In a previous study published in Science, researchers discovered how to regulate the movement of molecules so they can find and properly engage
with moving cellular receptors.
By mimicking the movement of biomolecules, synthetic materials can communicate
with cells.

A key innovation in the research is the discovery of how to control the collective movement
of more than 100,000 molecules in nanofibers.
Because cell receptors in the human body can move at extremely fast speeds — sometimes on timescales of milliseconds — they become moving targets
that are difficult to hit.

"Imagine dividing a second into 1,000 time periods
.
This is the speed
at which the receptor moves.
These timelines are so fast that they are difficult to grasp.

”

In the new study, Stupp and Kiskinis found that the nanofibers were tuned to contain the most active molecules, resulting in the highest degree of neuronal enhancement
.
In other words, neurons grown on a more dynamic coating—essentially scaffolds made up of many nanofibers—are also the most mature neurons, the least likely to aggregate, and have a stronger signaling capacity
.

"We think this is feasible because the receptor moves very fast on the cell membrane, and the signaling molecules of our scaffold also move very fast
," Stupp said.
"They're more likely to sync.

If the two dancers are out of sync, pairing won't work
.
Through very specific spatial contact, the receptor is activated
by the signal.
It's also possible that our fast-moving molecules enhance the movement of receptors, helping them clump together in favor of signaling
.
”

Neurons with ALS characteristics provide a new window into the study of the disease

Stupp and Kiskinis believe their mature neurons will provide insights into studying aging-related diseases and be better candidates
to test various drug therapies in cell culture.
Using this dancing molecule, the researchers were able to age human neurons vastly, allowing scientists to study the onset
of neurodegenerative diseases.

As part of the study, Kiskinis and his team extracted skin cells from an ALS patient and converted them into patient-specific iPSCs
.
They then differentiated these stem cells into motor neurons, the cell type
of this neurodegenerative disease.
Finally, the researchers cultured neurons on a novel synthetic coating material to further develop ALS signatures
.
Not only does this give Kiskinis a new window into studying ALS, these "ALNs" can also be used to test potential treatments
.

"For the first time
, we were able to see adult-onset aggregation of neuroproteins in stem cell-derived motor neurons of ALS patients.
It was a breakthrough for us," Kiskinis said
.
"It's unclear how the aggregation triggers the disease
.
" This is the answer
we hope to find for the first time.
”

It is hoped that in the future it will be able to treat spinal cord injuries and neurodegenerative diseases

In the future, IPSC-derived maturation-enhancing neurons could also be transplanted into patients with spinal cord injury or neurodegenerative diseases
.
For example, doctors can take skin cells from patients with ALS or Parkinson's disease, convert them into iPSCs, and then grow those cells on a coating to create healthy, powerful neurons
.

Transplanting healthy neurons into patients can 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 will be genetically matched to the patient, eliminating the possibility of
rejection.

"Cell replacement therapy is very challenging for diseases like ALS because motor neurons transplanted from the spinal cord need to project their long axons to the appropriate surrounding muscle sites, but may be more direct
for Parkinson's disease," Kiskinis said.
"In any case, this technology will be revolutionary
.
"

"It is possible
to take cells from patients, turn them into stem cells, and then differentiate into different types of cells," Stupp said.
"But the yield of these cells tends to be low, and achieving proper maturation is a big problem
.
" We can integrate our coatings into large-scale manufacturing of patient-derived neurons for cell transplantation treatments without immune rejection
.
”