As Madeline Lancaster lifts a clear plastic dish into the light, roughly a dozen clumps of tissue the size of small baroque pearls bob in a peachcolored liquid. These are cerebral organoids, which possess certain features of a human brain in the first trimester of development—including lobes of cortex.
“Copyright © 2015, All rights reserved MIT Technology Review; www.technologyreview.com”
Organoids are miniature, artificially grown organs of the human body. They consist of the same cellular types as the real organs and can form a three-dimensional self-organizing structure. There are organoids of the intestine, kidney, liver, inner ear, etc. but also tissue that resembles the developing human brain. These cerebral organoids or brain organoids stem from the self-organization of pluripotent neural stem cells that recapitulate the neural development of an embryo, including the early formations of the distinct cortical layers of the human brain.
With the creation of cerebral organoids, we are able to form neurological models of human brains that allow us to study diseases and developmental stages of an embryonic brain. The differences between the physiology of human and mammalian brains limit the framework of researching neurological disorders. Cerebral organoids may help us to overcome these issues. One fundamental aim of creating organoids is making them as similar as possible to the original. However, the closer we get to reach this aim, the more arises the question at what point a pile of nerve cells growing in a Petri dish becomes a brain that functions just like the one everyone of us has. Would it eventually start to think, feel, love and have dreams just like we do?
Let us stop at this point, take a step back and look how these cerebral organoids came into play and how they were created.
Broadly speaking, human pluripotent stem cells (hPSCs) are self-replicating stem cells that form amongst other things the nervous system and are characterized by their ability to differentiate into multiple cell types that reside in our brains. In the development of the human cortex they generate radial glial progenitor cells, which are similar to stem cells but more limited in their capacity to differentiate and divide. These radial glial progenitor cells, which are located in the ventricular zone, a temporary embryonic layer of tissue, in turn generate the neurons in the cerebral cortex and glia cells. In the assembly of the cerebral cortex the newly generated neurons migrate past their predecessor to form a more superficial layer, eventually leading to six distinct layers of the humans cortex, which are organized in an ‘inside-out’ manner, where the earliest born neurons reside in the deepest layer and the latest born neurons form the outermost layer. This results in a brain structure that is unique among mammals.
Madeline Lancaster, an American developmental biologist invented the cerebral organoids by a happy accident. She was trying to grow two-dimensional neural rosettes, a typical radial arrangement of embryonic neural stem cells used for research, but due to her lack of experience in tissue culture, the cells did not behave as she expected. They failed to stick to the bottom of the dish and started growing onto each other forming little floating balls, that vaguely resembled a brain tissue. Lancaster saw the potential in her alleged misfortune and published a paper in 2013, where she described these mini-brains, in which she and her team managed to develop structures of the cerebral cortex and human specific embryonic zones of the brain, launching a whole new branch of research of in vitro models of the human brain that offers tremendous possibilities. Previous studies have already modelled certain cortical tissues, but not like the one Lancaster has created. Her cerebral organoids had the unique organization of differentiating stem cells and stereotypical inside-out organization of discrete cortical layers.
The cells of a growing embryo are divided into three germ layers: endoderm, mesoderm and ectoderm. Each developing into various parts of tissue and organs. The nervous system is derived from the ectoderm. The procedure of the cultivation of cerebral organoids starts with generating neuroectodermal tissue from natural embryoid bodies, more precisely, two-dimensional colonies of human pluripotent stem cells (hPSCs). The hPSCs are then transformed into a three-dimensional cell culture by embedding them in droplets of a gelatinous protein mixture, a basement membrane matrix, to provide a scaffold for more complex tissue growth. In there, the stem cells float in a nutrient broth consisting of all the components necessary for the development of neurons. The gel droplets are subsequently transferred to a spinning bioreactor that improves the nutrient absorption. The implementation of a rotating bioreactor was the key step in cerebral organoid construction, as it allowed faster cell doubling and increased cell expansion. Different types of neurons and brain tissues are then forming rapidly. After just ten days the first neurons develop and after 20 to 30 days the cerebral organoids form complex heterogeneous tissues, which could survive up to ten months when maintained in the bioreactor.
But how does a stem cell “know” into which type of cell it has to differentiate?
Cell fate is controlled by several factors that influence the process of determination. The spatial location of the cells and temporal attributes of a neural progenitor can decide if it becomes a neuron or a glia cell. But also, extracellular factors and multiple interlinked cell signalling pathways with distinct signalling molecules like transcription factors (proteins that control the transcription of DNA) play a central role in cell fate. The exact mechanisms and stimuli of what makes a neural progenitor cell to differentiate into a specific type of cortical layer remain unknown. Further research of cerebral organoids has a lot of potential in supporting the investigations of those developmental mechanisms.
However, one of the biggest problems that hinders the evolution of cerebral organoids is the difficulty of cultivating a network of blood vessels within the organoids, which is essential for further growth. It means that the tissues reach a size limit and stop growing after approximately two months. Given that brain organoids have a very dense organisation that is highly dependent on exact cell connectivity between the neurons, providing each cell with the appropriate amount of oxygen, glucose and other nutrients in this organisation is almost impossible yet. Despite making immense progress in research, particularly in forming the three-dimensional structure of cerebral organoids, they are not yet miniature replicas of human brains. It is still unknown how important brain regions are formed in the anatomically correct places. Howard Fine, a neuro-oncologist from New York City, stated in this context: “Our cerebral organoids have almost all the major functional and neuroanatomical structures you’d find in a developing fetal brain, but their positioning is kind of random. If you were developing a whole body, your ear would be where your knee is.”
Recent work on two-dimensional cultures of neural stem cells has already produced neurons that are interconnected via synapses and are capable of firing action potentials. But with the implementation of 3D self-organizing cultures science has made an even greater step forward. Lancaster claims that human pluripotent stem cells can be used to produce organoids which “faithfully recapitulate the early periods of human embryonic (…) brain development on a cell-biological and gene expression level”. The organoids can already grow to the size of 4 millimetres replicating in vitro the in vivo development of the fetal brain. The study conducted by Lancaster et al. even demonstrated that distinct and interdependent brain regions in these mini brains exhibited neural activity via calcium oscillations, which is an element of synaptic transmission between neurons. Following the application of exogenous glutamate – a excitatory neurotransmitter – even more frequent calcium spikes were observed, indicating glutamatergic receptor activity. This increase in activity could subsequently be blocked by applying tetrodotoxin, a neurotoxin that also occurs naturally in some species of blowfish and is able to block voltage-gated sodium channels that are required for generating an action potential in a neuron. According to Lancaster the observed weakening of these calcium waves indicates that they were dependent upon neural activity in these organoids. Along with these achievements on a physiological level, immunohistochemical assays of the mini brains revealed areas reminiscent of cerebral cortex, choroid plexus, hippocampus, retina and meninges.
The possibilities emerging from these techniques are countless. Things that seemed impossible a few decades ago are now within reach, as scientist are now able to observe living human brains develop in a petry dish. Various drug compounds and genetic modifications can be tested on human tissues, making animals models almost obsolete in the future. Additionally, these mini brains can be grown from an individual’s stem cells, thus providing personalised organoids, that can enhance the accuracy of creating customized treatments and model for a wide range of diseases like brain tumours and Alzheimer’s taking into account interindividual differences. For instance, organoids enable researchers to display tumour development in real time. In comparison, visualizing brain tumours in mice requires the animals to be killed, which also stops the tumour from growing further, making it impossible to observe all specific stages of development of the cancer. Recently, the production of cerebral organoids from a person’s own stem cells has been approved by an institutional review board, which would allow incorporating a patient’s immune cells into these models, making them more realistic and in future allowing potential cell replacement therapies. And finally, the so-called holy grail of medical science could be reached by transplanting fully grown organs into humans, overcoming issues of compatibility and rejection by the immune system.
Giving these organoids blood vessels, immune cells and a more realistic structure remains a big challenge though, but the field of brain organoids is only five years old yet and still bears enormous potential. Despite [IK1] these limitations, cerebral organoids already are a highly valuable model system for medicinal research on diverse diseases like microcephaly, Alzheimer’s, schizophrenia or autism. The formation of brain organoids from human pluripotent stem cells might be one of the greatest discoveries in biomedical research. In combination with cell reprogramming technology and gene editing via CRISPR/CAS9 it allows us to reach frontiers that were unthought of a few decades ago. The possible fields of application are numerous and transplanting healthy functioning brain tissue into patients with severe lesions to critical brain areas now doesn’t sound like science-fiction anymore but rather something, which is just a matter of time.
Despite these great promises, the construction of cerebral organoids raises several urgent ethical questions about whether organoids, once able to function like a fully developed brain or in the process to do so, might also start to develop basic cognitive processes and eventually a kind of consciousness. The neurons in brain organoids are already capable of firing action potentials and form interconnections via synapses providing the basic framework of neural circuits. The closer scientists get to the actual developing brain in an embryo and the bigger the organoids grow and the stronger the interconnection between neurons via synaptical transmission manifests, the more likely it will be that neural circuitries might emerge. We should then ask ourselves whether cerebral organoids are or will be sentient and conscious one day.
Recently, a team of researchers led by neuroscientist Alysson Muotri of the University of California were growing hundreds of brain organoids to form cortical tissue, which in vivo includes regions that control cognition and interpret sensory information. Each organoid resembled a typical developing human brain in terms of their gene expression. They subsequently recorded the electrical activity in the organoids via electroencephalography (EEG) and were surprised by the results that indicated that these organoids were firing at a much higher frequency than other brain organoids they had previously created and weren’t specifically induced to grow cortical tissue. The mini brains displayed irregular patterns of EEG activity, that appeared like the chaotic burst of synchronized neural activity in real developing human brains at 25-39 weeks of age. Still, these intriguing findings do not prove that we are growing organoids that may be equal to brains of an almost newborn. Organoids are far from being comparable to a sentient and conscious human brain. Scientist are not able to cultivate each cell type that naturally occur in the cortex yet, further the cortex cells in organoids don’t connect to other artificial brain regions. Also, the issue of vascularisation within the brain tissue remains the biggest limiting factor in growing bigger and more complex organoids. Nevertheless, as the project of cerebral organoids further develops, ethical questions about researching on artificial brains that might be able to feel and think some day will be raised quickly. Interestingly, there is not even an agreement among scientist how to measure consciousness in real human brains, posing yet another challenge on how to measure it in organoids.
So, is it ethical to even continue this branch of research?
Cerebral organoids can be considered a breakthrough on multiple levels. In general, it is ethically more acceptable to experiment with artificially grown organs than with a complete organism, may it be a mammal or a human volunteer. No living being needs to be killed, damaged or put a risk in the process. Immunological personalised treatments with cerebral organoids will probably exceed the accuracy and reliability of traditional treatments. In the future the production of cerebral organoids could also be cheaper in terms of material and working hours than other forms of experimentation. All this leads to the impression, that the creation and use of cerebral organoids is highly desirable and that it even might be considered unethical not taking this opportunity. Still, at this stage cerebral organoids are too small and primitive and for now this endeavour remains a promising opportunity. But if we indeed find signs of basic sentiments or consciousness in the process of creating bigger and more complex cerebral organoids, then the ethical questions will become pressing.
Chen, H. Isaac; Song, Hongjun; Ming, Guo-Li (2019): Applications of Human Brain Organoids to Clinical Problems. In: Developmental dynamics: an official publication of the American Association of Anatomists 248 (1), p. 53–64. DOI: 10.1002/dvdy.24662.
Di Lullo, Elizabeth; Kriegstein, Arnold R. (2017): The use of brain organoids to investigate neural development and disease. In: Nature reviews. Neuroscience 18 (10), p. 573–584. DOI: 10.1038/nrn.2017.107.
Lancaster, Madeline A.; (11/30/2015). Growing mini brains to discover what makes us human [Video file]. Retrieved from https://www.youtube.com/watch?v=EjiWRINEatQ&t=9s
Lancaster, Madeline A.; Knoblich, Juergen A. (2014): Generation of cerebral organoids from human pluripotent stem cells. In: Nature protocols 9 (10), p. 2329–2340. DOI: 10.1038/nprot.2014.158.
Lancaster, Madeline A.; Renner, Magdalena; Martin, Carol-Anne; Wenzel, Daniel; Bicknell, Louise S.; Hurles, Matthew E. et al. (2013): Cerebral organoids model human brain development and microcephaly. In: Nature 501 (7467), p. 373–379. DOI: 10.1038/nature12517.
Lavazza, Andrea; Massimini, Marcello (2018): Cerebral organoids. Ethical issues and consciousness assessment. In: Journal of medical ethics 44 (9), p. 606–610. DOI: 10.1136/medethics-2017-104555.
Nowogrodzki, Anna (2018): How cerebral organoids are guiding brain-cancer research and therapies. In: Nature 561 (7724), p. 48-49. DOI: 10.1038/d41586-018-06708-3.
Purves, Dale (Hg.) (2008): Neuroscience. 4. ed. Sunderland, Mass.: Sinauer Assoc.
Reardon, Sara (2018): Lab-grown ‘mini brains’ produce electrical patterns that resemble those of premature babies. In: Nature 563 (7732), p. 453. DOI: 10.1038/d41586-018-07402-0.
Zhu, Z.; Huangfu, D.; (2013): Human pluripotent stem cells – An emerging model in developmental biology. In: Development, 140(4):705-17. DOI: 10.1242/dev.086165