(photo credit: INGIMAGE)
Healthy brains of newborns are usually wrinkled like walnuts, while those born without wrinkles suffer from “smooth brain syndrome,” which is connected to severe developmental deficiencies and a markedly reduced life expectancy. The gene that causes this syndrome recently helped Weizmann Institute of Science researchers to probe the physical forces that cause the brain’s wrinkles to form.
In their findings, reported Monday in Nature Physics, the researchers describe a method they developed for growing tiny “brains on chips” from human cells that enabled them to track the physical and biological mechanisms underlying the wrinkling process.
Tiny brains grown in the lab from embryonic stem cells – so-called organoids – were pioneered in the last decade by Profs. Yoshiki Sasai in Japan and Juergen Knoblich in Austria.
Prof. Orly Reiner of the Weizmann Institute’s molecular genetics department says that her lab, along with many others, embraced the idea of growing organoids.
But a researcher in her lab, Dr. Eyal Karzbrun, put a damper on her enthusiasm by pointing out that the sizes of the organoids they had obtained were far from uniform. Likewise, with no blood vessels, the insides did not have a steady supply of nutrients and started to die. Furthermore, the thickness of the tissue interfered with the optical imaging and microscope tracking.
Even before the paper’s publishing date, the scientific community showed great interest in this new approach to growing organoids. “It is not exactly a brain, but it is quite a good model for brain development,” said Reiner. “We now have a much better understanding of what makes a brain wrinkled or, in cases of those with one mutated gene, smooth.” The researchers plan to continue developing their approach, which they believe could lead to new insights into other disorders that are tied to brain development, including microcephaly, epilepsy and schizophrenia.
Karzbrun developed a new approach to growing organoids – one that enabled the group to follow their growth processes in real time: He limited their growth in the vertical axis, giving him a pita-shaped organoid – round and flat with a thin space in the middle. This shape enabled the group to image the thin tissue as it developed and to supply nutrients to all the cells. And by the second week of the tiny “brain’s” growth and development, wrinkles began to appear and then to deepen. Karzbrun noted: “This is the first time that folding has been observed in organoids, apparently due to the architecture of our system.”
A physicist by training, Karzbrun naturally turned to physical models for the behavior of elastic materials to understand the formation of the wrinkles. Folds or wrinkles in a surface are the result of mechanical instability – compression forces applied to some part of the material. So for example, if there is uneven expansion in one part of the material, another part might be forced to fold in order to accommodate the pressure. In the organoids, the scientists found such mechanical instability in two places – the cytoskeleton (internal skeleton) of the cells in the center of the organoid contracted and the nuclei of the cells near the surface expanded – or, he explained, the outside of the “pita” grew faster than its inside.
While this achievement was impressive, Reiner was not convinced that the wrinkles in the organoids were really modeling the folds in a developing brain. So the group grew new organoids, this time bearing the same mutations carried by babies with smooth brain syndrome. Reiner had identified this gene – LIS1 – back in 1993 and has investigated its role in the developing brain and in the disease, which affects one in 30,000 births. Among other things, the gene is involved in the migration of nerve cells to the brain during embryonic development, and it also regulates the cytoskeleton and molecular motors in the cell.
The organoids with the mutated gene grew to the same proportions as the others, but they developed few folds and the ones they did develop were very different in shape from the normal wrinkles. Working on the assumption that differences in the physical properties of the cell were responsible for these variations, the group investigated the organoid’s cells with atomic force microscopy, with the help of Dr. Sidney Cohen of the institute’s chemical research support department. By measures of elasticity, the normal cells were about twice as stiff as the mutated ones, which were basically soft.
Reiner concluded: “We discovered a significant difference in the physical properties of cells in the two organoids, but we observed difference in their biological properties as well.”