Human brain development illustration

Newswise — Possibly the most complex organ in the entire living world, the human brain has long been an object of fascination for researchers. However, studying the brain and in particular the genes and molecular switches that regulate and control its development is no easy task.

So far, scientists have worked with animal models, especially mice, but their findings cannot be directly transferred to humans. A mouse brain is structured differently and lacks the ridged surface typical of the human brain. Cell cultures have heretofore been of limited value in this area because cells tend to spread over a large area when grown on a culture dish; this does not correspond to the natural three-dimensional structure of the brain.

Mapping molecular fingerprints

A research group led by Barbara Treutlein, ETH professor at the Department of Biosystems Science and Engineering in Basel, has now taken a new approach to studying the development of the human brain: They grow and use organoids – millimeter-sized, three-dimensional tissue made up of so-called pluripotent stem cells can be grown.

Given these stem cells receive the right stimulus, researchers can program them to become any type of cell found in the body, including neurons. When the stem cells are aggregated into a small clump of tissue and then exposed to the appropriate stimulus, they can even self-assemble and form a three-dimensional brain organoid with a complex tissue architecture.

In a new study just published in NatureTreutlein and her colleagues have now examined thousands of individual cells within a brain organoid at different points in time and in detail. Their goal was to characterize the cells molecularly, i.e. the entirety of all gene transcripts (transcriptome) as a measure of gene expression, but also the accessibility of the genome as a measure of regulatory activity. They managed to present this data as a kind of map showing the molecular fingerprint of each cell within the organoid.

However, this method creates immense data sets: each cell of the organoid has 20,000 genes, and each organoid in turn consists of many thousands of cells. “This results in a gigantic matrix that we can only solve with the help of suitable programs and machine learning,” explains Jonas Fleck, a doctoral student in Treutlein’s group and one of the co-first authors of the study. In order to analyze all this data and predict gene regulation mechanisms, the researchers developed their own program. “We can use it to generate an entire interaction network for each individual gene and predict what will happen in real cells if this gene fails,” says Fleck.

Identify genetic switches

The aim of this study was to systematically identify those genetic switches that have a significant impact on the development of neurons in different regions of brain organoids.

With the help of a CRISPR-Cas9 system, the ETH researchers switched off a specific gene in each cell, a total of around two dozen genes simultaneously in the entire organoid. In this way, they were able to find out what role the respective genes played in the development of the brain organoid.

“This technique can be used to screen for genes involved in diseases. We can also look at how these genes affect how different cells develop within the organoid,” explains Sophie Jansen, also a PhD student in Treutlein’s group and second co-first author of the study.

Review of pattern formation in the forebrain

To test their theory, the researchers chose the GLI3 gene as an example. This gene is the blueprint for the transcription factor of the same name, a protein that attaches to specific sites in the DNA in order to regulate another gene. When GLI3 is switched off, the cellular machinery is prevented from reading this gene and transcribing it into an RNA molecule.

In mice, mutations in the GLI3 gene can lead to malformations in the central nervous system. Its role in human neuronal development was previously unexplored, but mutations in the gene are known to lead to diseases such as Greig cephalopolysyndactyly and Pallister-Hall syndrome.

By switching off this GLI3 gene, the researchers were able to both check their theoretical predictions and determine directly in cell culture how the loss of this gene affected the further development of the brain organoid. “We have shown for the first time that the GLI3 gene is involved in the formation of forebrain patterns in humans. This had previously only been shown in mice,” says Treutlein.

Model systems reflect developmental biology

“The exciting thing about this research is that you can use genome-wide data from so many individual cells to postulate what role individual genes play,” she explains. “What I find equally exciting is that these model systems, made in a Petri dish, really reflect the developmental biology we know from mice.”

Treutlein also finds it fascinating how self-organized tissue can develop from the culture medium, the structures of which are comparable to those of the human brain – not only on a morphological level, but also (as the researchers have shown in their latest study) on the level of brain gene regulation and pattern formation. “Organoids like this are really a great way to study human developmental biology,” she says.

Versatile brain organoids

Research into organoids from human cell material has the advantage that the findings can be transferred to humans. They can be used to study not only basic developmental biology, but also the role of genes in brain disease or developmental disorders. For example, Treutlein and her colleagues are working with such organoids to research the genetic cause of autism and heterotopia; in the latter, neurons appear outside of their usual anatomical position in the cerebral cortex.

Organoids can also be used to test drugs and potentially cultivate transplantable organs or organ parts. Treutlein confirms that the pharmaceutical industry is very interested in these cell cultures.

However, growing organoids requires both time and effort. In addition, each clump of cells does not develop in a standardized way, but rather individually. That’s why Treutlein and her team are working on improving the organoids and automating their manufacturing process.