Like many around the world, Professor Mriganka Sur’s lab at MIT’s Picower Institute for Learning and Memory has adopted young brain organoid technology, or “minibrains,” to study human brain development in health. and disease. By making a surprising discovery about a common practice in the process of growing complex tissue cultures, the lab has produced both new guidelines that may improve the technology, as well as new information about the important roles that a predominant enzyme plays in the natural development of the brain.
To make organoids, scientists take skin cells from a donor, trick them into becoming stem cells, and then cultivate them in a bioreactor, guiding their development with the addition of growth factors and other chemicals. Over the weeks, the stem cells become progenitor cells which multiply and then become, or “differentiate”, into neurons or other types of brain cells. Along the way, cells also migrate into the growing gout to take their place forming basic brain structures and circuits.
A beauty of organoids, therefore, is that, as cells in the culture grow and develop together, they simulate many basic processes that occur when real brains take shape. When cell donors have genes that cause the disease, the organoid from their cells mimics the underlying characteristics of the disease. The Sur lab uses organoids to study abnormal brain development in Rett syndrome, a devastating autism-like disease with a genetic basis.
A common practice among organoid culture labs is to improve cell viability by adding a small chemical molecule called CHIR 99021 to inhibit the activity of a ubiquitous natural enzyme called GSK3-beta. In the new study in PLOS ONE The team, led by Picower Fellow Chloé Delépine, confirmed that while different doses of CHIR 99021 can actually keep cells alive, they have opposite effects on organoid growth – low doses promote growth but high doses constrain it. and very high doses will stop it completely. This information alone has obvious implications for laboratories using different doses of CHIR 99021, but as Delépine’s team studied how these growth effects occur, it also helps clarify what the activity of GSK3-beta is. affects during brain development.
This is an important question. Other research groups, for example, have shown that disturbances in a signaling pathway involving GSK3-beta in the brain are associated with schizophrenia and autism. The new findings model how different levels of inhibition can affect development.
“It’s not just about increasing the viability of cells, it also changes other cellular processes such as division, differentiation and migration,” Delépine said. “Our idea was to test the effects of different doses and to better understand these mechanisms that we observed.”
Eight organoids are presented in three columns. They appear as irregularly shaped white spots. A scale marker suggests they are about 3mm wide.
Organoids showed different degrees of growth depending on the dose of CHIR 99021. Controls are on the left. Those in the middle received a low dose. Those on the right received a high dose.
Different doses, opposite effects
To conduct the study, Delépine’s team cultured organoids and, from day 15 to day 35 of their development, treated them with doses of either an inert control or 1, 10 or 50 micromolar of CHIR 99021 They then followed various properties of the cells by staining. for different molecular markers of these properties.
Immediately, she noticed major differences in organoid size depending on the dose received. Low dose organoids (1 micromolar) were 1.6 times larger than controls, but high dose organoids (10 micromolar) were 1.8 times smaller and very high dose organoids (50 micromolar) had it all. just stopped growing at the start of treatment.
The differences in growth were not due to cell death. As expected, since laboratories use CHIR 99021 to improve cell survival, the addition of CHIR 99021 improved cell viability and the effect was stronger for high dose treatments than for low dose treatments. But when the lab looked for markers of cell proliferation or division, they found that high-dose organoids had less while low-dose organoids had more than controls.
When the team looked at another cellular activity that could affect growth, they discovered a similar pattern. In high dose organoids fewer cells differentiated into neural progenitor cells needed to produce new neurons, while in low dose organoids proliferation increased. As a result, high dose organoids had fewer neurons.
In addition, the low dose of CHIR 99021 caused more migration of newborn neurons to places necessary for structural organoid development than the control treatment, suggesting that a slight inhibition of GSK3 beta contributes to migration. increased.
Delépine noted that the growth promoting effects of low doses or the growth limiting effects of high doses are neither “good” or “bad”. These are just levels of culture control. The new study provides more information on how to use CHIR 99021 to achieve the desired goal.
“It really depends on the purpose of the organoid research you’re doing,” she said. “Depending on what you want, different doses of this molecule can help you get a different phenotype.”
And in natural brains, the study suggests that GSK3-beta likely plays a key role in the proliferation of progenitor cells, their differentiation into mature brain cells, and the propensity of those cells to migrate.
Besides Delépine and Sur, the other authors of the journal are Vincent Pham and Hayley Tsang.
The National Institutes of Health, the Simons Foundation Autism Research Initiative, and the JPB Foundation supported the research.