A fascinating tale of brains in a dish

The promise and peril of stem cell derived cerebral organoids; a complex, interconnected 3D model system of the human brain

Immunohistochemistry of a cerebral organoid

In recent years, one might have stumbled across various sensationalist headlines of “scientists created breakthrough mini-brain in a dish”, putting the prospect of finding cures for diseases like Alzheimer’s or microcephaly just a few years in the future. As with many sensationalist stories, you might not have heard much since.

So where is the field standing? And why is nobody talking about it?

Stick around, it is a fascinating story!

The origins of the initial journalistic hype came from a newly developed model to study the brain outside a body, so-called “stem cell derived human brain organoids”. In short, organoids are just a collection of stem cells grown over long time in a 3D culture system; where they would form complex structures resembling organs from liver to pancreas to lungs and brains. To get stem cells to commit to a certain direction of development (“differentiation”), scientists would adjust environmental conditions (oxygen/nutrition gradients and so forth) or put growth hormone “cocktails” in the culture media to simulate certain environments.

For initial “mini-brains”, human embryonic stem cells were first prepared in flat 2D dishes and developed to embryoid bodies and then subjected to neural induction media, prompting differentiation to neuroectodermal tissue.

Neuroectodermal tissues were then maintained in three-dimensional culture and embedded in droplets of Matrigel to provide a scaffold for more complex tissue growth. These Matrigel droplets were then transferred to a spinning bioreactor to enhance nutrient absorption (Fig. 1a). This method led to rapid development of brain tissues, which we termed cerebral organoids, requiring only 8–10 days for the appearance of neural identity and 20–30 days for defined brain regions to form. — Lancaster M. et al., Nature, 2013
A long complicated culture process gives the cells enough time and space to form more complex structures

Amazingly, Madeline Lancaster and colleagues from the Austrian Academy of Science, together with collaborators from UK based renowned Welcome Trust Sanger Institute and the University of Edinburgh found that their cerebral organoids would not only develop into a bundle of neuronal cells, but that various discrete, interdependent brain regions would arise out of their model system.

While thoroughly fascinating on its own, the authors then continued to investigate if their model system could be applied in a disease context. It is well established that microcephaly, a medical condition where the brain does not develop properly, can have many causes, from inherited chromosomal abnormalities to acquired disruptive injuries like ischemic stroke to viral infections (e.g. Zika virus). Basically a lot of things can go wrong in developing something as complex as the adult brain.

In their study, the authors focused on microcephaly caused by a single mutated gene, as discovered by sequencing of a microcephaly patient. They took some skin cells from that patient, retransformed them into pluripotent stem cells, and then used their 3D culture model to develop these patient-derived cells into brain organoids. To their surprise, the patient derived stem cells (carrying a single mutation in a gene) behaved completely similar to control pluripotent stem cells during the first days of their program. However, once they switched to neural induction media, the mutant cells would not develop under the same conditions. After investigation the researchers found out that the genetic mutation in the patient causes premature differentiation while failing to maintain progenitor cells, leading to overall smaller neural tissues, recapitulating the reduced brain size seen in the patient.

Lancaster et al.’s 2013 study started a gold rush for neurobiologists to apply cerebral organoid model systems to their respective research areas, and the hype is still growing.

Enter 2017, human brain organoids have become so prominent and interesting for research that Georgia Quadrato and colleagues from Harvard and MIT took up the enormous task of analyzing the gene expression of over 80,000 individual cells isolated from 31 human brain organoids.

Large-scale single cell sequencing and clustering techniques allow scientists to look into what their little stem cell have become during organoid development

For people outside the field, single-cell sequencing is not a cheap or easy method, the scope of 80.000 cells would have been impossible just 5 years ago. In any case, better methods yield better research, and Georgia Quadrato and colleagues really went out of their way to provide a comprehensive overview on brain organoid development. With several computational clustering and visualization techniques, the authors mapped all cells according to similarities, to identify and classify the diversity of brain organoid’s cellular makeup.

Human brain organoids contain subclasses of forebrain and retinal cells, as well as cellular subtypes from photoreceptor and glia cells to amacrine and epithelial cells.

Their comprehensive study provided for the first time a systematic overview on what many scientists already suspected; human brain organoids grow more diverse the longer they are allowed to progress. Furthermore, these bundles of increasingly diverse cells would start interacting in complicated ways, beginning to self-organize and form complex 3 dimensional structures resembling distinct brain regions. All in a dish.

Let that sink in for a moment.

We found that although some cell types were present at both three and six months, some appeared only at six months. Of note, at three months the forebrain cluster did not contain the population of putative callosal projection neurons, which in vivo are born in large numbers during late corticogenesis. Similarly, Müller glial and bipolar cells, which are normally generated at late stages of retinal development, were absent from the three month dataset. -Quadrato G. et al., Nature, 2017

Now is the time we go from fascinating to mind-blowing. Given their findings of enhanced neuronal maturity in their brain organoid system, the authors started investigating whether spontaneously active neuronal networks would be present as well. A neuronal network is defined as active neurons who are functionally connected and can transmit a so-called spike, an electrochemical signal, over several synapses to other neurons. They used silicon microelectrodes to record electrical pulses in the oldest (8 months) organoids at different recording sites and detected median firing rates of 0.66 Hz, which could be suppressed by chemical inhibition of voltage-gated sodium channels (= receptors necessary for neurons to fire). In contrast, they were unable to measure any spikes in only 4 months old organoids.

Finally, since the authors discovered photoreceptor cells (usually part of the eye’s retina) within their single-cell sequencing data, they wondered if they could modulate electrical spike rates by simply exposing these cells to light (~530nm wavelength, saturated green color). Out of all their older organoids, the four that had developed light-responsive units would attenuate their firing rates when hit by the green light, as well as upregulate an activity-dependent gene named FOS, supporting the notion that these organoids indeed harbor photosensitive cells.

Human brain organoids develop spontaneous neural networks which can be modulated by sensory stimulation

Yet any good science comes with the caveat of opening more questions than it answered. Quadrato et al.’s extensive study just opened the door into a new world of complexity, as well as provided a sneak peak of the troubles ahead: Reproducibility, the great peril of biological sciences. The sheer diversity of cell types and structural subclasses between brain organoids was enormous, a surprising discovery given that these organoids were all derived from genetically identical stem cells and developed largely under the same culture conditions.

If it holds true that even subtle changes in culturing condition have such profound effects on individual differences in organoid diversity, scientists will have a bona fide chaotic system at their hands. Maybe a system too chaotic to control for. Time will tell.

At least Quadrato and colleagues seem optimistic:

The diversity and maturation of cell types generated, the robustness of the neuronal networks, the presence of structural traits of mature neurons and the possibility of using sensory experience to modulate neuronal activity collectively suggest that, beyond modelling early events of progenitor biology, these 3D brain organoids have the potential to model higher-order functions of the human brain, such as cellular interactions and neural circuit dysfunctions related to neurodevelopmental and neuropsychiatric pathologies. — Quadrato G. et al., Nature, 2017

I hope this answered our original question of where the field is standing;

Scientists are still hyped about growing human brain organoids in vitro, as these systems allow to study brain development and disease in a new and interesting way. Furthermore, the potential of stem cells to self-organize and form complex brain-like structures given the right conditions is absolutely baffling and poses more questions than it answers. Which makes it hard to cover for journalists.

In general, when journalists first hype and then drop a hot research area, it usually boils down to two things:

  • Overestimation of short-term application and neglect of long-term potential

Human brain organoids cannot single-handedly provide cures for neuropathologies or explain the brain in an easy fashion, but they might teach us more than we can anticipate. Just because the short-term promise does not pan out as hoped, we should not be ignorant on long term potential. Remember, also the internet started small.

  • Complexity increases as research progresses, demanding more nuanced understanding and making coverage harder

At least this second point journalists and scientists have in common, the brain is confusing for everybody. Brain organoids get so much more complicated the longer they are allowed to progress, who can really grasp all their depths? And then try to do the coverage justice?

After all, isn’t there some irony in asking our brain to understand itself when looking at a mirror in a dish?

My brain certainly has to think about that reflection.

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