Summary: Brain organoids help researchers map the molecular, genetic, and structural changes that occur during brain development.
source: ETH Zurich
The human brain is perhaps the most complex organ in the entire living world and has long been an intriguing subject for researchers. However, studying the brain, especially the genes and molecular switches that regulate and direct its development, is not an easy task.
So far, scientists have proceeded to use animal models, primarily mice, but their results cannot be transferred directly to humans. The mouse brain is formed differently and lacks the wrinkled surface typical of a human brain. Cell cultures have so far been of limited value in the field, as cells tend to spread over a large area when grown on a culture dish; This is inconsistent with the normal 3D structure of the brain.
Molecular fingerprint mapping
A group of researchers led by Barbara Treutlein, professor of ETH at the Department of Biosystems Science and Engineering in Basel, has now taken a new approach to studying the evolution of the human brain: They grow and use organoids – millimeter-sized, three-dimensional tissue that can be cultured from what are known as pluripotent stem cells.
Provided these stem cells receive the right stimulation, researchers can program them to become any type of cell in the body, including neurons. When stem cells are assembled into a small ball of tissue and then exposed to the appropriate stimulus, they can even organize themselves and form a three-dimensional brain organelle with a complex tissue structure.
In a new study just published in temper natureTreutlein and her colleagues have now studied thousands of individual cells within a brain organoid at different time points and in great detail.
Their goal was to characterize cells in terms of molecular genes: in other words, the sum of all gene transcripts (transcripts) as a measure of gene expression, but also accessibility of the genome as a measure of regulatory activity. They were able to represent this data as a kind of map that shows the molecular fingerprint of each cell within the organelle.
However, this procedure generates massive data sets: each cell in the organelle contains 20,000 genes, and each organelle in turn is made up of many thousands of cells.
“This results in a giant matrix, and the only way we can solve it is with the help of appropriate software and machine learning,” explains Jonas Flick, a doctoral student in the Treutlein group and one of the study’s co-authors. To analyze all this data and predict the mechanisms of gene regulation, the researchers developed their own program.
“We can use it to create a complete interaction network for each individual gene and predict what will happen in real cells when that gene fails,” says Flick.
Identification of genetic keys
The aim of this study was to systematically identify those genetic switches that have a significant impact on neuronal development in different regions of the brain’s organelles.
With the help of the CRISPR-Cas9 system, ETH researchers selectively turned off one gene in each cell, a total of about twenty genes simultaneously in the entire organoid. This enabled them to learn the role of the genes involved in the development of the organisms in the brain.
This technique can be used to examine the genes responsible for the disease. In addition, we can look at the impact of these genes on how different cells develop within the organelle,” explains Sophie Janssen, also a doctoral student in the Treutlein group and second co-lead author of the study.
Verification 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 cleaves to specific sites on DNA in order to regulate another gene. When GLI3 is turned 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 abnormalities 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 Greg’s cephalopolysyndactyly and Pallister Hall syndromes.
Silencing this GLI3 gene enabled the researchers to verify their theoretical predictions and to determine directly in cell culture how the loss of this gene affected the further development of the brain organ.
We have shown for the first time that the GLI3 gene is involved in the formation of forebrain phenotypes in humans. This has previously been shown only in mice, Treotlin says.
Model systems reflect evolutionary biology
“The exciting thing about this research is that it allows you to use genome-wide data from many individual cells to compute the roles that individual genes play,” she explains. “What’s exciting to me is that these model systems made in a petri dish really do reflect evolutionary biology as we know it from mice.”
Treutlein also finds it fascinating how a culture medium can give rise to self-organizing tissues with structures similar to those of the human brain—not only at the morphological level but also (as the researchers demonstrated in their latest study) at the level of gene regulation and pattern formation.
She points out, “Organisms like this are really an excellent way to study human developmental biology.”
Versatile brain organelles
The advantage of research on the organelles that make up the substance of human cells is that the results can be transmitted to humans. They can be used not only to study basic developmental biology but also to study the role of genes in diseases or developmental brain disorders.
For example, Treutlein and her colleagues are working with organoids of this species to investigate the genetic cause of autism and hypertrophy. In the latter, neurons appear outside their usual anatomical location in the cerebral cortex.
Organisms can also be used to test drugs, possibly to grow transplantable organs or parts. Treutlein confirms that the pharmaceutical industry is very interested in these cell cultures.
However, organelle growth takes time and effort. Moreover, each block of cells develops individually and not in a uniform manner. That’s why Treutlein and her team are working to improve memberships and automate their manufacturing process.
About this brain mapping news
author: Peter Roig
source: ETH Zurich
Contact: Peter Rueegg – ETH Zurich
picture: Photo credited to F. Sanchís Calleja, A. Jain, P. Wahle / ETH Zurich
original search: open access.
“Inference and disruption of cell fate regulation in human brain organellesWritten by Barbara Treutlin et al. temper nature
Inference and disruption of cell fate regulation in human brain organelles
Self-regulating neural organoids transplanted from pluripotent stem cells combined with single-cell genetic techniques provide opportunities to examine the gene-regulatory networks underlying human brain development.
Here we obtain single-cell transcript and chromatin data accessible over a dense time course in human organs covering neuroepithelial formation and modelling, brain region and neurogenesis, temporally identifying dynamic regulatory regions and specific brain region.
We have developed Pando – a flexible framework that includes multifactorial data and predictions of transcription factor binding sites to infer a global network of gene regulation describing the evolution of organoids. We use the combined genetic perturbation with single-cell transcriptome readouts to assess transcription factor requirements for cell fate and state regulation in organelles.
We find that some factors regulate the abundance of cell fates, while others influence the states of neurons after differentiation. We demonstrate that the transcription factor GLI3 is required for the establishment of cortical fate in humans, summarizing previous research conducted in mammalian model systems.
We measure transcriptional and chromatin accessibility in normal or perturbed GLI3 cells and identify two distinct GLI3 systems that are central to focal fate decisions: one that regulates dorsal centrosome patterning with HES4/5 as direct targets of GLI3, and one that controls nodal diversity prominent in later development. .
Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.