Small Brain Big Potential

A scientist arranges petri dishes containing mini brains such that they read “mini” from left to right.
A scientist arranges petri dishes containing mini brains such that they read “mini” from left to right.

Introducing, to the field of neuroscience, mini brains. Also known as brain organoids, mini brains have forever changed neuroscience research [1]. At first blush, you might picture mini brains as just that—shrunken models of the brain sitting in your head right now. Imagining tiny brains floating around in a petri dish may seem preposterous, but broadly, you would be correct. Science is always striving to find the newest research techniques and shiniest technologies. With the introduction of mini brains, the potential to reshape the future of neuroscience is now within reach.

Mini brains are lab-produced neurological structures composed of human stem cells. To understand the theory behind mini brain technology, it is first essential to understand how stem cells function. Stem cells are special cells that have the power to transform into multiple types of body cells. This process of cells choosing their specialized function is known as differentiation. Once stem cells differentiate, they can no longer change their specialized functions [2]. Throughout the body, there is a wide variety of stem cells, some of which have the ability to become any type of cell, such as Induced Pluripotent Stem Cells (iPSC), and some of which have a more limited fate selection [2]. Unlike other varieties of stem cells, iPSCs have no functional limitations and have the power to become any body cell [3]. Consequently iPSCs are used to develop in vitro samples—that is, samples outside of a living organism—of neural cells to create mini brains [4].

The most useful quality of pluripotent cells is that researchers can manipulate them to become whichever type of cell they seek to study. For example, in a 2021 study at the Sloan Kettering Institute for Cancer Research, researchers differentiated stem cells into central nervous system cells in order to study their response to viral infection [5]. Stem cells are unique and powerful tools and have been used for research in fields ranging from cancer to autoimmune diseases such as diabetes [3]. Stem cells have also made their way to the field of neuroscience as they provide researchers with a controlled way to study the two types of nervous system cells: signaling cells, known as neurons, and supporting cells, known as glia. These cells are the building blocks of neural structures, and through their manipulation, researchers have the power to model any neural structure they so desire [2]. Thus was born the mini brain.

Mini brains are complex, three dimensional (3D) in vitro, tissue models composed of neurons and glial cells [6]. As opposed to traditional two dimensional (2D) culture models that are simply cells on a plate, mini brains are physical collections of tissue that exist beyond the plate’s plane [7]. However, they are not exact replicas of functioning human brains: you can think of human brains as desktop computers and mini brains as their cell phone counterparts. The foundational neural cells that comprise mini brains are derived from the differentiation of stem cells.

Stem cell-derived neural cells are used to generate the 3D mini brain models through a variety of techniques. One example is through culturing the derived neural cells on low adhesion cell surfaces. This culturing method, in which cells are unable to stick to the surface of the culturing plate, coupled with constant agitation, causes the cells to clump into tissue models or the physical mini brains [4]. The mini brain models are roughly the size of a grain of sand [4]. These models have comparable cellular composition to normal brains, but they are not exactly anatomically correct—just as cell phones are not exactly the same as computers.

Although our current understanding of brain development and function is a product of animal models, such models do not fully capture the complexities of the human brain [1]. The use of stem cells in constructing mini brain structures significantly reduces the possibility for variation between the cells of mini brains and the cells of functional human brains. The relative similarity between mini brains gives researchers a proxy to investigate human brain activity in response to medications or genetic mutations, aside from animal models [1]. For example, in the past, the physiological abnormalities associated with Alzheimer’s have been corrected in mouse models of the disease. However, this has not been possible yet in humans [8, 9]. Although we do not know the exact reasoning behind this discovery, the difference in effectivity between the treatments of Alzheimer’s in humans and in mice suggests a disconnect between the neural structure and function of human and mouse brains. It also demonstrates the limitations of animal models in regard to human brain function. Although mini brain models lack input from other body systems, such as immune signals, mini brains still offer a precise method of studying human tissue in a manner that more accurately captures cell development and interactions.

The following is a diagram mapping how mini brains were utilized for studying Rett syndrome. First, an individual diagnosed with Rett syndrome was recruited, then the individual’s skin cells were extracted, then the cells were used to derive iPCs, these iPCs contributed to the generation of mini brains, and finally these mini brains were tested with candidate drug therapies.
The following is a diagram mapping how mini brains were utilized for studying Rett syndrome. First, an individual diagnosed with Rett syndrome was recruited, then the individual’s skin cells were extracted, then the cells were used to derive iPCs, these iPCs contributed to the generation of mini brains, and finally these mini brains were tested with candidate drug therapies.

In practical application, scientists are able to use mini brains to study particular parts of a neuron, such as the dendrites and axons, and observe the influence of large scale disease and drug effectiveness [4]. Because the development and construction of mini brains are completely laboratory-controlled, researchers have the ability to tag certain cells. This means that neurons can be labeled with dyes or markers that allow researchers to visualize the morphological or structural organization processes that occur throughout mini brain development [4]. This labeling process, where particular protein sequences are inserted into genes to mark cells, allows researchers to monitor those cells of interest for changes in structure and reactions to treatments [10]. The method can be further applied to the greater examination of neuronal organization. Overall, mini brains provide a model for drawing greater specificity and insight into the internal structure and function of brain cells.

In theory, mini brains seem like the optimal method of modeling human brain behavior. From drug and disease modeling to greater clarification of neural structure, mini brains represent a precise and novel means of exploring the intricacies of the human brain. However, while they are revolutionizing the way in which scientists study and conceptualize the brain, some technical and ethical limitations still hinder the widespread use of this technology. A 2020 study conducted by Arnold Kreigstein et al. at The University of California San Francisco shed greater light on the true precision of mini brain technology. In this study, researchers examined certain active genes in the cells of lab-generated mini brains as compared to normally developing brains. Of the 235,000 cells collected from mini brains and the 189,000 cells collected from normally developing brains, researchers found multiple discrepancies in gene expression when the same genes were examined from both samples via single-cell RNA sequencing. Significant differences in gene expression showed variation in the expression of the HOPX gene, a marker of glial cells in the outer brain [11]. This finding led to the conclusion that mini brains are not as accurate a representation of normal neural developmental processes as once thought [11]. More specifically, the study found that the stem cells undergoing a transformation into neurons or other mature cells in mini brain structures do not fully complete the developmental process; instead, they are less molecularly diverse and lack the glial cell maturation signature of typical neurons [11]. Consequently, the mini brains constructed from these immature cells do not fully represent a typical brain’s organizational structure [11].

Additionally, many of the artificially derived cells in mini brains showed signs of metabolic stress, rendering them perpetually exhausted. This stress was primarily attributed to the upregulation, or greater expression, of the metabolic stress genes, ARCN1 and GORASP2 [11]. Cells in typical functioning brains are not stressed in this way, and as such, are as energetic as one might be at the beginning of a race. Mini brain cells, in contrast, were shown to be tired, in the way one may feel at the end of a race. This discrepancy in stress levels changes cell activity as more energy is dedicated to relieving the stress than to typical cell function.

Notably, when the mini brain cells were transplanted into mouse brains, their stress levels decreased. They behaved more like typical cells with improved identity and function [11]. This evidence shows that mini brains should not be dismissed; the tendency of cell quality to improve when transplanted into a mouse suggests that it is possible to overcome both the incomplete maturation and metabolic stress-related limitations of mini brains. Though this finding contributes to the theory that mini brains do not reflect true brain function, the accuracy of mini brains is improving, and these models are still important representations of small areas of the human brain.

Lastly, there remains an ongoing ethical debate concerning the potential development of consciousness in these lab-grown brains. A study released in 2019 showed that lab-generated mini brains produce coordinated electrical waves that resemble the brain activity of developing fetuses [12]. While mini brains are incredibly far from developing any such consciousness as defined by humans, this evidence raises the concern of the evolution of consciousness in organs used specifically for research. It is an important point to address as the technology continues to develop. Just as it is unethical to conduct invasive research on living people, would it also be unethical to conduct studies on non-corporeal but conscious tissue entities? While something to consider, this worry raises the paradox between ethics and the ongoing pursuit of scientific research—to constantly strive for the most accurate data and research models. As mini brain technology continues to progress and elements such as continuous blood flow and sensory signal input are incorporated into the model, where do we draw the line between sentient humans and pure research models?

Despite these technical and ethical limitations, mini brains and the conclusions that can be drawn from them remain important. While it is crucial to recognize and understand the constraints of such technologies, mini brains are the first biologically accurate testable models of human brains. Also, mini brains still represent a breakthrough in the breadth of possibilities for human brain research, drug testing and disease modeling being only a few.

A chief example of the application of mini brains to clinical research is the 2020 study conducted by Alysson R. Muotri et al. at the University of California San Diego examining Rett syndrome. Rett syndrome is an uncommon, currently incurable autism spectrum disorder that impairs brain development. The syndrome is believed to be caused by mutations on the X-linked MECP2 gene, which encodes a regulatory protein necessary for typical brain development [6]. Much like how a car will run abnormally with the wrong tire, mutations in the MECP2 gene cause changes to cell shape, gene expression patterns, and pathway function, amongst other factors, that consequently alter the “running” of overall neural activity [6]. As a result, Rett syndrome is often characterized by difficulty with movement, speech, and breathing [4].

In their study, Muotri and colleagues sought to identify drugs effective for the treatment and management of Rett syndrome, using mini brains as a primary model. They began by deriving iPSCs from the skin cells of previously diagnosed Rett syndrome patients, thereby preserving the MECP2 mutation. The resultant stem cells were then used to construct mini brain models which mimicked the brains of actual Rett syndrome patients. Armed with these biological representations of affected brains, Muotri and colleagues were able to test the viability of 14 drug candidates for Rett syndrome treatment and produce precise and accurate results. Their findings showed that two medications, Nefiracetam and PHA 543613, successfully reversed the synaptic and network pathway alterations caused by MECP2 mutations [6]. While a groundbreaking discovery, this identification is still relatively new, and, currently, Nefiracetam and PHA 543613 are undergoing further research for use in humans [6]. In this study, mini brains were essential to the precise identification of new drug candidates for the treatment of Rett syndrome. Use of a different technique, such as an animal model, would not have produced results nearly as accurate due to this method’s failure to mimic the cellular interactions of true brains as precisely as mini brains. While not perfect, mini brains provided a new, and potentially more accurate, model for Rett syndrome pathology and development.

It is important to keep in mind that this is only one example of the application of mini brain technology in neuroscience research. The ability of mini brains to adopt the genetic and neurological state of any condition opens the door to endless possibilities. From evaluating potential drugs to modeling the physiology of aging neurons, scientists now have the rare opportunity to closely observe and specify complex brain function [1]. The discoveries made from Muotri’s study only represent the beginning of the scientific progress made possible by mini brains.

Mini brains may also soon make their way beyond scientific research and into the medical field itself. In fact, in the next ten to 20 years, the currently hypothesized prospect of “personalized organoids” may be realistically implemented. Personalized organoids are derived directly from the cells of patients and can be coupled with effective drug testing to yield personalized insight into the drugs most suited to individual people [1]. This method holds the potential to greatly enhance the effectiveness of clinical treatment and only represents one possible application of mini brain technology outside of biomedical research.

Although mini brains have their limitations, their growing popularity suggests that the technology will only improve in the future [1]. With continuous use, the specificity, function, and structure of this model is bound to more closely resemble the activity of true brain function. Rett syndrome treatments, an Alzheimer’s cure, and personalized organoids— these possibilities are just the tip of the iceberg. Though they are “mini,” these brains have giant potential.

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