In a Petri dish, beneath a layer of nutrient-rich medium and faint microscope light, a small
cluster of neurons stirs. These cells, taken from the brain of an axolotl, extend delicate arms toward each other, seeking connection. Nearby, Bostan watches a computer screen
flicker with pulses of light—calcium signals, a sign that the neurons are communicating.
With feathery gills curling from its head and an eternal smile, the axolotl (Ambystoma
mexicanum) has long been a darling of science. Unlike most amphibians, axolotls retain their larval features throughout their lifespan, a condition known as neoteny. Axolotls are also extraordinary regenerators: while most animals can heal minor wounds, axolotls can restore entire limbs, spinal cord segments, heart tissue, and even portions of the brain. This regenerative capability makes them valuable for studying tissue repair and neural
regeneration. But for decades, research on brain regeneration in axolotls was at a
standstill.
Even though scientists have known about axolotls regenerating brain tissue since 1964, it was hard to study because we weren’t able to raise axolotl neurons in cell cultures —until now. In the Öztürk Lab at Istanbul Medipol University, researchers Sevginur Bostan and Safiye Serdengeçti are working together to answer one question: if axolotl brains can regenerate, why can’t ours? They work in complementary areas—Bostan studies neuron-astrocyte interactions, while Serdengeçti investigates perineuronal nets (PNNs), specialized
extracellular structures that support neurons in mammals.
One of the many reasons axolotls can regenerate their limbs while humans can’t is that when mammals have spinal cord injuries, astrocytes, a type of glial cell (cells that support the nervous system), form a protective scar around the wound. Although this scar stabilizes the spinal cord, it also blocks neurons from reconnecting and regenerating. Axolotls’ glial cells, on the other hand, are more plastic and have the potential to become neurons, allowing them to rebuild the damaged circuitry. At the same time, the surrounding space, or extracellular matrix, stores a “memory” for repairing the damaged tissue and provides structural support for the new neurons. These processes are not well understood because, before Bostan, no one had been able to culture either axolotl neurons or the supporting glial cells.
Translating the axolotl's regenerative capabilities into reproducible in vitro models has been a significant challenge. It was also an issue that Bostan didn’t think she would solve. Bostan initially pursued a master’s degree in molecular biology before switching to neuroscience at Istanbul Medipol University. There, she worked on developing new methods for growing neurons to study neural activity, particularly through microelectrode arrays. Growing neurons on lab plates was already second nature. So, when she was invited to explore regeneration in axolotls, a species she admits she “didn’t have a deep knowledge of”, she decided to give it a shot.
Traditional mammalian neuronal culture protocols proved inadequate for axolotl cells from
the moment Bostan collected them. Brain matter consists of a wide variety of material, which is best removed from cultures. While this debris is easily degraded in mouse brain cultures, the team had to centrifuge and filter out the axolotl cells from a cocktail of debris. Another difference is the growing process itself. Neurons typically have to be grown on a surface. But while mammal neurons stick to a surface and start re-extending their axons in a matter of hours, axolotl neurons take weeks. One of Bostan’s biggest breakthroughs came from using cloning cylinders. By penning up the cells within small polystyrene rings, she forced them to stick to the surface. Another difference in culturing axolotl cells was having to lower the temperature to 21°C, far below the 37°C standard used in most mammalian cell cultures. In these cultures, the two were able to record neurons extending neurites, the branching arms that connect cells. They also observed the neurons forming synapses and firing in coordinated bursts of calcium activity, patterns reminiscent of early mammalian neural networks.
These cultures not only survived but also matured functionally. The neurons grew complex
branching structures, called neuritic arbors, and formed synaptic connections, as shown by
immunocytochemistry. Surprisingly, some cells expressed both neuronal and glial (support
cell) markers. Why this happens in axolotls isn’t yet clear, but it may reflect greater plasticity in their nervous system. Early tests have already revealed intriguing activity: axolotl neurons show spontaneous calcium signaling similar to mammalian neurons, suggesting that fundamental principles of neural circuits are conserved despite evolutionary distance. These results provide strong evidence that the axolotl’s regenerative abilities arise from intrinsic cellular properties and can be studied in vitro. One important piece remains missing: pure populations of glial cells. So far, these support cells are largely absent, likely lost during early preparation steps.
To prevent the brain cells from clumping excessively, the team used DNase from Biomatik to remove sticky DNA on the surface of each cell and adjust the pH. Biomatik produces
custom gene synthesis, custom proteins, and antibodies for researchers. Biomatik has
delivered 72,000+ custom-made products, in addition to catalog products and ELISA kits.
Due to supply chain issues and closures in partner companies, the original formulation of
DNase from Biomatik is now discontinued.
Although the axolotl project was primarily led by Bostan (soon to be Dr.), it was also
completed in large part thanks to Safiye Serdengecti, a neuroscientist with a background in psychology. Specifically, she studies the perineuronal net (PNN), a dense, sugar-rich mesh that wraps around certain brain cells like scaffolding. In mammals, these nets stabilize the brain’s wiring as we age, locking circuits in place after critical periods of learning.
Using live imaging and molecular probes, Ms. Serdengeçti has shown that even in mammals, the PNN isn’t fixed. In response to large bursts of activity, it can be dynamically reshaped by enzymes called matrix metalloproteinases (MMP). The possibility that the PNN could be temporarily loosened has far-reaching implications. In schizophrenia, postmortem studies have found that PNNs are abnormally structured in brain areas tied to thinking. In Alzheimer’s, their gradual loss may underlie the unraveling of memory.
While neural regeneration and plasticity are often studied separately, they share common
molecular pathways and cellular mechanisms that become apparent when comparing
regenerative and non-regenerative systems.
In axolotls, elements in the extracellular matrix encourage regeneration, making way for the neuron buds, and lack many of the inhibitory components produced by mammalian glial scars. This permissiveness may be actively maintained by MMP activity that prevents the accumulation of inhibitory matrix components. The hybrid neuronal-glial phenotype observed in Bostan's axolotl cultures suggests that these cells may retain a degree of plasticity that mammalian glia lack. Mammalian astrocytes typically respond to injury by becoming reactive and contributing to glial scar formation, but it’s hypothesized that differences in glial cells may account for differences in regeneration. Understanding the molecular differences between these responses could reveal strategies for reprogramming mammalian glia to adopt a more pro-regenerative phenotype.
Going forward, Bostan and Serdengeçti are looking to delve into the gaps in axolotl neural
research. They want to find more mechanisms behind the regenerative ability of salamanders, and even ways to genetically modify those cells for further study. One stunning difference the two noticed is that axolotl cells are particularly difficult to transfect -the process of changing an organism’s DNA using viruses. This difficulty, Bostan speculates, may be due to the cells’ autophagy pathways, which allow the cell to remove infected components. It is also a system associated with regenerative processes.
As this research progresses, it may ultimately lead to transformative therapies for conditions ranging from spinal cord injury to neurodegenerative diseases, bringing us closer to a future where the human nervous system can, like the axolotl's, recover what was lost. The salamander does not grow up. But perhaps it has something to teach us about how to begin again.