The first time the three children’s brains grew, things went so completely off the rails that they developed severe epilepsy and autism. Now biologists have used stem cells from these patients, who have a devastating disorder called Timothy syndrome, to grow their brains a second time — in miniature, in a lab dish.
The scientists reported in Nature on Wednesday that the mini-me cortices reprised the abnormal brain development so faithfully that the research might point the way toward preventing or treating the rare disorder.
Producing mini-Timothy-syndrome-brains-in-a-dish is only one of the remarkable new advances in the exploding science of “cerebral organoids,” miniature, three-dimensional human brain-like structures. These lab creations, scientists hope, will mimic severe psychiatric and neurological diseases such as schizophrenia, autism, and Alzheimer’s much better than mouse brains (the current go-to choice) do, revealing what goes wrong and offering a testing ground for experimental treatments.
But ever since cerebral organoids were first created from stem cells in 2013, they have ignited an intense ethical debate, including about whether they can suffer, feel pain, or be conscious — and whether they have human rights.
While that may seem far-fetched, a second study, also published in Nature Wednesday, described growing hundreds of cerebral organoids from the stem cells of healthy people for up to nine months and longer in special bioreactors. The mini-brains developed dozens of kinds of brain cells, from those that give rise to neurons in the cerebral cortex to those that connect the right brain and the left brain. And they linked up into working circuits — not producing thought or emotion, but pulsing with the same kind of electrical activity that enables human brains to do both.
“This shows that the approach has much greater potential than we ever imagined,” said Juergen Knoblich, of the Institute of Molecular Biotechnology in Austria, a pioneer in creating cerebral organoids who was not involved in either study. “They’ve shown that if you keep [the mini-brain] growing for a long enough time, it will generate the whole repertoire of cells we see in the human brain.”
Together, the new papers “give us unprecedented ways of looking at a human brain and its development,” said biologist Timothy O’Brien, of the University of Minnesota, who has also produced brain organoids from stem cells and was not involved in the new work. A key advance in both studies, he said, was “uncovering more layers of complexity, showing that these things really do look like human brains.”
In the Timothy syndrome study, scientists at Stanford University School of Medicine began with human cells called fibroblasts, donated by three patients. Using now-standard techniques, they put those cells through a biological time machine, returning them to an embryo-like stem cell state. By adding the proper nutrients, the researchers could not only spur these stem cells to grow into brain cells — done for more than a decade — but to form little balls.
Each is about 1/16 inch across and contains more than 1 million cells. Called cortical spheroids, they differ from cerebral organoids in that the former mimic specific regions of the brain, such as the front, rather than many sections.
Stanford’s Dr. Sergiu Pasca and his colleagues hoped the spheroids would reveal what goes wrong in Timothy syndrome. One batch mimicked a region deep in the brain, and another mimicked the cortex, the crevassed outer layer where thinking occurs. In fetuses, neurons from deep in the brain migrate and connect to neurons in the cortex, forming circuitry that supports thought, judgment, planning, and other higher-order functions.
After fusing the two cortical spheroids — cortex and deep layers — Pasca and his colleagues watched the neurons for days, seeing that those from the deep layer were terrible migrants. They stutter-stepped and stopped and started as if unsure whether or where to move. Yet they migrated more than neurons in normal brains, as if trying to make up for their confusion by barreling forward willy-nilly.
The spheroids, Pasca said, “help us see how brain development goes awry in patients with the different mutations linked to Timothy syndrome.” (Stanford has filed for a patent on generating brain-region-specific spheroids.)
Drugs called calcium-channel blockers, which are commonly used to treat high blood pressure, produced normal neuron migration in the mini-brains, Pasca said, apparently by patching the defect that makes the neurons too jumpy. Even if further studies show the drugs restore normal neuronal migration, however, a big question looms: Will giving the drugs after birth, when the brain’s basic wiring diagram is set, be too late?
“We don’t know,” Pasca said. “But there may be a window of opportunity later,” in infancy or even childhood, to correct brain circuitry.
Earlier research has already produced cerebral organoids that developed distinct regions such as the hippocampus, motor regions, and visual areas, with electrically active neurons. When created from the stem cells of a patient with microcephaly, the brains-in-a-dish resembled that often-fatal condition; those created from cells of patients with severe autism indicated that out-of-control neuron growth is the underlying cause of that disorder.
But the second study on Wednesday went even further. Scientists at Harvard University grew their brain organoids, also from stem cells, longer than ever before: nine months or more. Growing in flasks in spinning bioreactors that kept them doused with nutrients, the organoids developed more mature and more diverse neurons than previous mini-brains. Sophisticated genetic tests on individual cells showed a diversity of neurons like that in real brains.
There were dozens of kinds: cells that give rise to support cells called glia, neurons that inhibit others, and corpus callosum neurons that connect a brain’s hemispheres. “It’s quite impressive that an organoid developing in a flask can make a great diversity of neuronal types,” said Paola Arlotta of Harvard and the Broad Institute, who led the study. The longer an organoid developed, the more types of cells it had.
And the neurons functioned. They sprouted tiny projections, called dendritic spines, which receive signals from other neurons in a circuit, and six out of the seven organoids that grew for at least eight months formed active neuronal networks, spiking with electrical activity. They “connect with each other, forming circuits, and once they’re connected, they can synchronize their activity,” Arlotta said, potentially mimicking “higher-order functions of the human brain.”
Bioethicists are just beginning to grapple with the implications of that. Questions such as whether and when brain organoids might become sentient are “important,” Arlotta said, “and we need to discuss these issues as a community. Not just as scientists, but all of us.”
The largest mini-brains-in-a-dish are only 4 millimeters across — roughly the size of a sea slug or jellyfish brain — and, Minnesota’s O’Brien said, “a tiny, tiny fraction of the human brain. You do see some neural circuits forming, but none that are anywhere near the size needed for sentience, and they are not nearly complicated enough to feel pain.”
Developing better mimics of the human brain, he added, “will take a lot more time, but maybe less time than we think.”