TUESDAY, 19 DECEMBER 2023Have you ever lived in a city for so long that you witnessed it changing over the years? Streets got damaged and repaired, old shops went out of business and new ones opened up. I bet you can even picture that run-down cafe in the corner getting replaced by a fancy bar with queues of youngsters lining up at the weekends. Now imagine this dynamic city inside an opaque snowball and ask yourself, how could we study and understand those changes now? Some may suggest cracking it open, and some may suggest finding a way to make the snowball transparent. What would you suggest if the globe is someone’s brain and the intricate network of the city is made up of cells and tissue? Over the years, doctors and neuroscientists have tried to tackle this problem. Brain injuries and related pathologies affect millions of people every year and yet the brain is still a great enigma to us, partially due to its inaccessibility.
In some rare situations, doctors have been able to directly study functioning human brains. For instance, Dr Penfield applied local anaesthetic to his epileptic patients, removed part of the skull and stimulated certain areas while the patients stayed awake. This allowed the patients to report what they were experiencing when the local stimulations were applied. Through this method, Dr Penfield was able to understand the role of some brain regions and how they modulate our experiences and actions.
Alternatively, scientists can study post-mortem brains. Nevertheless, these are often extremely damaged and only provide a static picture of the last stage of a disease. What is really interesting to scientists is the ability to monitor the processes and events that are happening in the brain in real-time. Being able to study the developmental processes of the brain, and how neural networks change over time is critical for scientists to understand the mysteries that lead to the many functions and dysfunctions of the brain.
FROM MICE TO FISH TO HUMANS
The development of animal models has also allowed us to peer into the brain. We have all heard about lab mice, but in fact, there has been a wide range of animals used in brain research - from pigs and monkeys, to flies (Drosophila), fish (D. rerio), and worms (C. elegans). Each of these models has its advantages and disadvantages depending on the goal of the research. These models can be used to study the molecular mechanisms that drive the fundamental processes in the brain, such as synapse formation and network plasticity. They are also used in behavioural studies, in which we can see how animals react to certain cues and environments, and then link their behaviours to brain activity and molecular pathways. We can also genetically modify animals to replicate human pathology. This is done by introducing specific mutations associated with a human disease into the animal’s gene. Nevertheless, we have hardly ever been able to faithfully replicate human diseases. This is due to the fact that replication of the disease in an animal model relies on the extent of our knowledge of the disease and its causes. In many cases, the pharmaceuticals developed to treat a disease show great efficacy in animal models, but fail in humans. This is because either the animal protein or gene targeted by the drug does not exist in humans, or the animal drug target is distinct from the human version. Indeed, we must not forget that there are some evolutionary differences between us and other animals. In fact, the best way to study a human brain is by studying a human brain.
GOING BACK IN TIME
This brings us back to the original question of how to study a human brain. While it is certainly not ethical to remove someone’s brain to study, new technologies have found a new way to get around this issue. What if the brain did not belong to anyone, and was in fact, grown in a laboratory? Although we are not quite there yet, this field of research has been growing in recent years. Welcome to the 21st century!
Nowadays, scientists can collect cells from people’s skin and de-program them to a pluripotent state. Pluripotency is the ability that some cells have to give rise to any other cell type that we find in the body. Normally we only find these cells in the early embryonic states and we lose most of them later on. So for scientists to obtain them, they have to get some adult cells (normally skin), put them in a time machine, and send them back to their embryonic state. This “time machine”, in fact, is called the Yamanaka Factors, which are a group of proteins that activate certain genes needed for cells to become pluripotent. The resultant cells out of the ‘time-machine’ are called induced pluripotent stem cells (iPSC). In addition, we can culture iPSCs in different media (any liquid that supports cell growth) and differentiate them into any type of cell in the body. For example, if we want to study Parkinson’s disease, which affects the dopaminergic neurons of the Substancia Nigra region of the brain, we can obtain skin cells from the patients, revert them to iPSC and then differentiate them into that specific type of neuron. This allows us to have millions of neurons in our flask growing in 2D and behaving in a similar way as they do in the brain. A great advantage of this approach is that these patient-derived iPSCs would maintain the genetic background of the patient of origin. This would therefore, better mimic the individual characteristics of the disease in the patient (such as the degree of aggressiveness).
Despite these advantages, this approach has some limitations. For instance, these cells can only be kept in culture for a limited amount of time. Furthermore, they don’t recapitulate the complex structure and cellular heterogeneity in the brain. In other words, we are trying to mimic a 3D world on a 2D platform. Thankfully, 3D cultures have been developed to overcome some of these issues. Spheroids are small iPSC-derived 3D cultures. Owing to their 3D morphology, these cultures can mimic the brain’s architecture better. Inside the spheroid, we can obtain diverse regions and layers with different cell types, just as we would have in a human brain. Furthermore, the cells in a spheroid are surrounded by other cells in all directions, which helps to enhance the processes of adhesion and communication, and therefore, better mimics the reality of a human brain. Finally, another advancement in 3D cultures is that they can be kept for a longer time, allowing the cells to form more connections of higher complexity and allowing us to study them for longer.
Another emerging interest in recent years has been the extracellular matrix (ECM). The ECM is the space in between the cells, the non-cellular component present in all organs. It is formed by an intricate network of molecules linked together into a structurally stable composite. In the past, researchers thought that the ECM was a passive structure, but it is now evident that it has a very active role, not only in contributing to the mechanical properties of a tissue but also in cell attachment, cell migration and communication. By embedding the 3D cultures in matrices, it could allow us to model the human brain environment. Some of these cultures have already been used to study diseases, such as in brain cancer, epilepsy and bipolar disorder. Within the matrix we might be able to find novel drug targets that allow the development of treatments for these pathologies.
Despite the recent advances in the field, there is still a lot to be done. One limitation of these models is the non-existence of a vascular system within the 3D culture. In fact, once the brain spheroids reach a certain size, there will not be sufficient nutrients and oxygen flux inside the sphere, and so the core will become necrotic.
In recent studies, many researchers have started to come up with different ideas to surpass this limitation, but further improvements are needed. Currently, we have a broad range of tools and models that allow us to study the brain in many ways. This is a fast developing field filled with new and exciting discoveries. There is no doubt that with every new implementation that goes beyond the current limitations, we will be one step closer to obtaining brain cultures that resemble the human brain better. With this, we are one step closer to understanding this enigmatic organ and having better platforms to test drugs for brain-related diseases.
Anna Pujol Castiblanque is a Darwin College PhD Student studying Glioblastoma heterogeneity at the Mair lab and Markaki lab. Prior to coming to Cambridge, she completed her undergraduate in Biotechnology at the University of Barcelona and her MSc in Brain and Mind Sciences at UCL and Sorbonne. Anna is particularly interested in patient derived human models for the study and modelling of brain pathologies. Illustration by Josh Langfield.