Stem Cell-Derived Neurons Navigate to Form Connections in the Injured Brain

Some parts of the body can recover from injury fairly rapidly. The cornea, for example, can heal from minor scratches within a single day. The human brain, however, is not one of these fast-healing tissues or organs. Adult brain cells are stable and last for a lifetime— barring trauma or disease—while some cells lining the gut last only five days and must be continually replaced.

Scientists would like to use stem cell therapy to boost the brain’s ability to regenerate following damage resulting from concussion or stroke. To date, such treatments have been stymied due to injury-related changes in the brain, as well as with difficulties integrating regenerated cells into existing brain circuits to restore functions such as memory retention or motor skills.

Scientists headed by a team at Sanford Burnham Prebys Medical Discovery Institute and Duke-National University of Singapore (NUS) Medical School now report the results of testing a regenerative therapy derived from human stem cells. Their studies showed that when transplanted into mice, the cells matured, integrated into existing circuits and restored function. By tracing the cells and sequencing their gene expression patterns, the researchers also revealed how transplanted cells find where they need to go and form connections with the nervous system. The studies showed that the cells contain their own intrinsic codes for navigation, and once they become neurons, this code instructs the cell to send its axons to a specific area of the brain.

Headed by said Su-Chun Zhang, MD, PhD, the Jeanne and Gary Herberger Leadership chair in neuroscience and the director of and professor in the Center for Neurologic Diseases at Sanford Burnham, the researchers reported their findings in Cell Stem Cell, in a paper titled “Transcriptional code for circuit integration in the injured brain by transplanted human neurons.” In their paper the team concluded, “Our finding opens a promising strategy for treating neurological diseases through promoting regeneration and neural transplantation.”

“The human brain has a limited regenerative capacity,” the authors wrote. Transplantation of neural progenitor cells (NPCs) offers a promising approach to replace the lost neurons and reconnect the damaged circuits, but there are challenges. “… how the grafted neurons find and make functional connections with their targets in the mature injured brain remains unclear,” they continued. “This makes neural transplantation therapy less predictable, especially for diseases of the cerebral cortex that affect diverse neuronal subtypes and multiple neural circuits, including the long-distance corticospinal tract (CST).”

One challenge facing regenerative medicine strategies for stroke and other forms of brain damage is the lack of a nurturing environment. Whereas the developing brain is a welcoming and instructive place for stem cells forming neurons and wiring the nervous system’s circuits, therapeutic cells arriving after a stroke find more hostility than hospitality.

“… injury leaves an open cavity surrounded by glial scars, resulting in a hostile microenvironment that severely limits the survival and integration of transplanted neurons, preventing effective structural repair and reconstruction of the damaged neural circuitry,” the investigators further noted.

“In the adult brain after a stroke, you see the formation of a cyst, a cavity that is filled with all sorts of inflammatory molecules, so it is a bit like the therapeutic cells are swimming in a dangerous swamp full of threats,”  Zhang further explained. “If that wasn’t enough, scar tissue surrounds the cavity to protect the brain from further damage, but it also forms a barrier against any potential regeneration.”

Some cell therapy strategists try grafting new cells next to the damaged region of the brain where it is easier for the cells to survive and grow. The goal is to eventually reestablish circuits by bypassing the damaged region. Zhang feels that this trauma needs to be healed rather than side-stepped to reach the potential benefits of regenerative medicine. “Following a stroke, the damaged lesion is often very large and presents an immense challenge to efforts to functionally reconnect the brain to the brain stem and spinal cord.”

Zhang and the research team sought to span this gap by developing a method to support the survival of therapeutic cells grafted directly into the harsh environment of the stroke cavity. Using a mixture of small molecule drugs and structural proteins, the scientists found that when transplanted into the injured mouse brain, the human embryonic stem cell-derived cortical neurons succeeded in surviving and growing to fill the damaged region. “The cortical NPCs were transplanted into the lesion cavity of ischemic stroke models … In total, we successfully performed cortical neuron transplants in 90 mice,” they wrote. Immunohistochemical analysis showed that 90% of transplanted cells expressed the neuronal marker NeuN, indicating differentiation into neurons.”

Zhang added, “Once transplanted cells can survive and become neurons, then we started asking whether those neurons can break through the scar tissue and grow functioning nerves by making new connections and reconstructing the disrupted circuits.”

While the researchers had proven it was possible to transplant cells and grow new neurons, they knew it would be of little benefit if they didn’t form the correct kinds of connections. Were they rebuilding bridges that had been demolished, or creating new bridges to nowhere?

After conducting three-dimensional reconstruction of the transplanted neurons, the scientists observed that the patterns of long, spiny projections neurons use to form connections in the nervous system resembled the patterns seen in normal neurons populating the pathway between the cerebral cortex and spinal cord. “We found that different types of transplanted neurons found their own partners even in the complicated context of the mature brain environment,” said Zhang. “They still can find their targets in a very specific manner.”

The scientists sought to better understand the navigational abilities of these regenerated neurons. They used a genetic barcode to label and trace the transplanted cells. This data was combined with the results of sequencing the transplanted cells’ gene expression profiles. “Single-nucleus RNA sequencing (snRNA-seq), in combination with barcoded retrograde tracing, revealed that transplanted cortical neurons showed subtype-specific projection and integration,” they wrote.

“We revealed that each cell type has its own code and, once the cells become neurons, this code tells each cell to send its projections or axons to different parts of the brain and spinal cord,” noted Zhang. “It’s the first time this striking phenomenon has been reported, and it is significant because it basically tells us that if we have the right types of transplanted cells, they already know where to go and what to do to repair what has been lost.”

The scientists used machine learning to identify four subtypes of neurons that develop from transplanted therapeutic cells. Each subtype has a distinct expression of genes known to guide the growth of axons, which explains why most neurons of a particular subtype send axons to form circuits with the same brain region. The research team also validated how axonal projection patterns are affected by transcription factor proteins that modify gene expression. They tested stem cells modified without a transcription factor called Ctip2 (CTIP2 knockdown; CTIP2-kd).

These transplanted cells’ projection patterns varied significantly from those with the factor, with more axons seeking to form connections with the hippocampus and amygdala. “The drastic change in projection patterns by the grafted cells with CTIP2-kd highlights the regulatable properties of the grafted neurons in pathfinding and circuit integration even in the adult brain,” the scientists stated. “…  the transcriptome of the transplanted cortical neuron subtypes may serve as identity tags to predict their projections and connectivity, providing a guideline for selecting appropriate neuronal cell types for targeted circuit reconstruction.”

Zhang concluded, “By learning more about these subtypes of transplanted neurons, we may be able to predict their projections and connectivity in order to select appropriate neuronal cell types for targeted circuit reconstruction in patients. It opens a promising future for cell therapy to help the millions of people that suffer from stroke and other devastating neurological conditions.”