The usage of Brain organoidsDeveloped in the laboratory from pluripotent human stem cells (capable of forming any type of tissue in the human body), it opens up new possibilities for studying various disorders and diseases that affect the human brain.
Researchers at the Institute of Molecular Biotechnology (IMBA), the Austrian Academy of Sciences and the ETH in Zurich (Switzerland) have developed a method that makes it possible to comprehensively test the effect of multiple cells in parallel and at the single-cell level mutations related to Autism Spectrum Disorder (ASD) within Human brain organoids. The results of the work are published today in the journal Nature.
Molecular biologist Jürgen Knoblich from IMBA, co-author of the work, is one of the world pioneers in the development of this type of organoid. As he tells SINC, his group has been working with them for more than a decade. “They are a great alternative to animal models and are particularly suitable for simulating processes that occur in humans but not in most animals.”
Organoids are a great alternative to animal models to simulate processes that only occur in the human brain
To develop, the human brain relies on processes unique to our species that allow us to build a complexly layered and connected cortex. These unique processes also make neurodevelopmental disorders more likely in humans. For example, many genes associated with a high risk of developing ASD are critical for the development of the cortex.
The complexity of the human brain
Although clinical studies have demonstrated the causal relationship between several genetic mutations and autism, it is not yet clear what causes these mutations Defects in brain development and due to the unique nature of human brain development, animal models are of limited use.
“Only a human brain model can replicate the complexity and special features of our brain,” emphasizes Knoblich.
“Recent research has revealed a growing number of aspects that differ greatly, for example between the human brain and that of the mouse,” explains the expert. That’s why we thought it was important to ask what the genes involved in autism do to him.”
Those from Knoblich and Barbara Treutleinfrom ETH Zurich and co-author of the study, allows us to examine a complete set of important transcription regulation genesassociated with autism, within a single brain organoid.
In this system that was called CHOOSE (CRISPR-human organoids-scRNA-seq)Each cell in the organoid carries at most one mutation in a specific ASD gene.
As Knoblich clarifies, they used the CRISPR Cas9 gene editing scissors “to modify genes thought to be involved in autism.”
Track the effect of each mutation
Using CHOOSE, the team was able to track the effects of each mutation at the single-cell level and record the developmental trajectory of each cell. “This high-throughput method allows us to systematically inactivate a list of disease-causing genes. As the organoids carrying these mutations grow, we analyze the impact of each on the development of each cell type,” he explains. Chong LiFirst author of the study and postdoctoral researcher in Knoblich’s group.
The CHOOSE tool makes it possible to detect the consequences of any mutation associated with ASD in a single experiment, thus drastically reducing analysis time
Knoblich, for his part, explains to SINC: “The characteristic and sensational thing about our method is that we do not ‘look’ for mutations, but rather ask ourselves in parallel what each of the already known mutations does in the human brain.” “We found an effect for every single one of them and it is this complete description of all genes and their consequences that makes our approach so unique.”
The tool makes it possible to “see the consequences of each mutation in a single experiment, drastically reducing the analysis time compared to other methods.” In addition, we can continue to benefit from a hundred years of scientific literature on disease-causing genes,” explains the Austrian scientist .
Quantitative bioinformatics and machine learning
Enormous amounts of data have been generated by mutating multiple genes in parallel and tracking their effects. For their analysis, Treutlein and his team at ETH Zurich used methods from quantitative bioinformatics and machine learning.
“Using this high-throughput single-cell expression data, we were able to quantify whether a particular cell type is more or less common due to a particular mutation. We were also able to identify groups of genes affected by each mutation. Comparing all mutations allows us to reconstruct the phenotypic landscape of these genetic disorders related to autism,” explains Treutlein.
Thanks to the CHOOSE system, the authors found that the 36 gene mutationsalready known to be associated with a high risk of ASD, lead to cell type-specific changes in the developing human brain.
In addition, they identified critical transcriptional changes regulated by common networks Gene regulatory networks (GNRs)., for its acronym in English). “A GRN is a set of molecular regulators that interact with each other to control a specific cellular function,” explains Li.
“We show that some cell types are more vulnerable than others during brain development and we identify the networks that are most vulnerable to autism-related mutations,” adds the lead author.
Knoblich points out that using this method, they found that “the genes that cause autism have some common molecular mechanisms.” However, these can have very different effects on different cell types.
There are cell types that are more susceptible to mutations that lead to ASD, particularly some neural progenitor cells, the cells that generate neurons.
According to Li, “there are cell types that are more susceptible to mutations that lead to autism, especially some neural progenitor cells, the cells that generate neurons.” This is true to the point that the pathology of autism could already occur in one early phase of brain development“suggesting that certain cell types will require more attention in the future when studying genes associated with ASD,” he says.
With stem cells from two patients
To confirm whether these findings are relevant to human diseases, the team collaborated with clinical researchers at the Medical University of Vienna and generated brain organoids from brain samples. Stem cells from two patients. Both had mutations in the same gene associated with autism.
“The organoids created by these two volunteers showed pronounced developmental disorders associated with a specific cell type. We then validate these observations in vitro “Comparison of the structures of these organoids with prenatal MRIs of the brain of one of the patients,” says Knoblich.
The observations showed that the organoid data agreed well with clinical observations, he adds.
Knoblich emphasizes that his results “provide a great reference for other researchers analyzing the disorder autism.” “In the future, researchers who come across a specific gene will be able to quickly search through our results.”
Additionally, the team highlights the versatility and portability of the CHOOSE system. “We expect that our technique will be widely used beyond brain organoids to study different genes associated with multiple diseases,” says this expert.
Li, C., Fleck, JS, et al. “Single-cell brain organoid screening identifies developmental disorders in autism.” Nature2023.