The scientist flicked on a laser, filling the rat’s brain with blue light. The rodent, true to its past two weeks of training, scampered across its glass box to a tiny spout, where it was duly rewarded with a drink of water. From the outside, this would appear to be a pretty run-of-the-mill neuroscience experiment, except for the fact that the neurons directing the rat to its thirst-quenching reward didn’t contain any rat DNA. Instead, they came from a human “mini-brain” — a ball of human tissue called an organoid — that researchers at Stanford University School of Medicine had grown in a lab and implanted in the rodent’s cortex months before.
The experiment — part of a study published Wednesday in Nature — is the first describing human neurons influencing another species’ behavior. The study also showed that signals could go the other way; tendrils of human neurons mingled with the rodent brain cells and fired in response to air rustling the rats’ whiskers.
The advance opens the door to using such human-rodent chimeras to better understand how the human brain develops and what goes wrong in neurological and psychiatric conditions such as schizophrenia, autism, and epilepsy. When the Stanford scientists implanted organoids grown from the cells of patients with a severe genetic brain disorder, they could watch the neurons develop abnormally with unprecedented clarity.
“This paper really pushes the envelope,” said neuroscientist Tomasz Nowakowski, of the University of California, San Francisco, who uses brain organoids in his research on neurodevelopmental disorders but was not involved in the new work. “The field is desperate for more experimental models. And what’s really important about this study is it demonstrates that brain organoids can complete their maturation trajectory when transplanted. So it really expands our toolkit for asking more nuanced questions about how genetic mutations lead to behavioral disorders.”
In biology, there is perhaps nothing more maddeningly difficult to study than the early development of the human brain. Acquiring tissue samples from that timeframe is virtually impossible. And so for decades, scientists have had to rely on indirect clues gathered from experiments on animals, like mice and monkeys. But the human brain is so evolutionarily distinct that those insights have not yielded much in the way of effective treatments for many behavioral disorders, which scientists believe are likely anchored in features unique to the human brain.
In the last 10 years though, new methods for coaxing stem cells to differentiate into neurons and growing those cells into 3D structures the size of a lentil have revolutionized scientists’ ability to study neurodevelopmental processes.
Mini-brains, brain balls, cerebral organoids, human spherical corticoids — whatever you call them — can trace their short, explosive history back to 2013, when developmental neurobiologist Madeline Lancaster, then at the Austrian Academy of Sciences, discovered that by growing stem cells in a salty-sweet supportive gel, the cells would self-organize into small, spherical masses of functioning brain tissue. Not long after, labs all over the world began growing up their own colonies of brain balls and applying different chemical prods and pushes to coax them to mature further.
In 2015, Stanford’s Sergui Pasca produced spheroids that could spark electricity, making them the first that neuroscientists could study functionally. A few years later, a group at UC Davis Medical Center published the first results of brain balls that could bleed, opening up avenues for studying strokes and other diseases of the brain’s vasculature. And the more like real organs they got, the more useful these blobs became for teasing apart the complex inner workings of the brain.
But there’s only so much scientists can learn in a test tube or gel matrix. In those environs, cells at the center of the spheroids get cut off from oxygen and nutrients as soon as they grow more than a few millimeters across. That limits their ability to develop functional neuronal networks — the connections and cellular crosstalk required to receive and process information — and thus their utility as a model for complex behaviors and neurological diseases.
Even Pasca, whose lab has reported some of the longest-lived brain organoid cultures at more than 800 days, noticed that neurons in a dish don’t grow as large as their real-life counterparts. Which is why he and his colleagues began searching out friendlier surroundings where the temperature is just right and there’s always a fresh flow of salt, sugar, and oxygen, like, say, inside the skull of a warm-blooded lab animal.
For this latest study, Pasca’s group chose two- to three-day-old rat pups, developmentally equivalent to a human infant — the time when brains are growing quickly and neurons are reaching out to form new connections. Into these animals, they transferred human organoids resembling the cerebral cortex. Some 80 percent of the implants took.
Within weeks, cells from the rats began to move into the organoids, building blood vessels that supported their growth alongside the animals’ own. From these organoids, millions of new neurons sprouted, sending out axons and wiring into circuits throughout the rats’ brains, including deep into the thalamus, a region responsible for relaying sensory signals such as touch and temperature. Six months later, about one-third of the brain hemisphere that received the transplant was made up of human cells.
Pasca’s group isn’t the first to have attempted an interspecies mind-meld. In 2018, scientists at the Salk Institute reported the first successful implant of human cerebral organoids into the brains of adult lab mice, where they survived for up to three months and even tapped into the animals’ neuronal circuitry. Later efforts by a group at Kyoto University repeated this feat in monkeys. But by doing their experiments in very young rats whose cortexes are not yet saturated with synapses, Pasca and his colleagues found that the human neurons easily integrated into the animals’ rapidly expanding circuitry, which provided them with the stimulation they needed to push past previous developmental barriers.
Analysis of the human neuron’s electrical properties showed “they’re much more mature than what we’ve done before in vitro,” Pascal said at a briefing for reporters on Monday. “They’re also much larger — they grow about six times larger than an equivalent neuron growing in a dish.”
That advance allowed them to see in much greater detail what goes wrong in the developing brains of children who develop severe epilepsy and autism due to a devastating genetic disorder called Timothy Syndrome. Pasca’s lab previously made mini-Timothy-syndrome-brains from the cells of patients, and in a lab dish, saw that their neurons jumped around haphazardly, unsure of where to migrate.
After growing for five to six months inside the brains of rats, those differences were even more stark. The Timothy neurons were smaller and stunted — they didn’t send out nearly as many dendrites, which foster connections with nearby neurons. As a result, the side of the rats’ brains with the Timothy transplant had much less, and much less organized, electrical activity.
“When you look in vivo, there’s a very clear difference between patient cells and control cells that you can literally see by eye,” Pasca said.
His group is now working to apply this method to study organoids created from patients with other neurological diseases, and hopes one day to use it as a drug-testing platform. Also on the to-do list: sticking even more sophisticated proto-brains inside the skulls of young rodents, where their development can go even further.
In a paper published in 2020, Pasca’s group showed that nestling together organoids representing three different tissues — skeletal-muscle, cerebral cortex, and spinal cord — created something that mimicked voluntary movement control. Zapping the brain end of this mega-blob, which Pasca dubbed an “assembloid,” caused the muscle end to twitch. He and his colleagues have generated organoids representing a dozen distinct brain regions, including deep regions of the central nervous system where inhibitory neurons are born.
Now Pasca’s group has begun to engineer assembloids with different ratios of excitatory and inhibitory neurons. By growing them inside the brains of young mice, they hope to be able to answer questions about the developmental roots of epilepsy, schizophrenia, and other psychiatric conditions believed to be caused by an imbalance between excitatory and inhibitory neurons.
In-Hyun Park, a stem cell biologist at Yale School of Medicine, has for years been making his own fusions of brain organoids to study how inhibitory neurons migrate during brain development. In a dish, his organoids produce a lifelike mix of excitatory, inhibitory, and support cells, but they don’t develop the kind of functional connections critical to understanding if the cells are behaving as they should. It looks like a brain, but does it talk like a brain?
To answer that, he said his team had been thinking about transplanting their organoids into lab mice, but they’d been reluctant — worried that taking over such a large part of the host’s brain would cause problems for the animal. But the work from the Stanford group has eased those fears. “They showed in a really powerful proof of principle that human neurons can functionally connect to the host brain,” Park said. “It’s a really important finding for the field.”
Neurosurgeons like Ben Waldau, of the University of California, Davis, agreed that the advance is a valuable one that will surely draw more researchers to work with organoids, and called the study “impressive.” But as someone interested in someday using organoids not just as a research tool, but to actually treat people who’ve lost brain function due to stroke, he noted that there’s still a lot of room for improvement.
Pasca’s organoids didn’t form any of the six layers that a normal human cortex has. And their integration into the rat brain wasn’t perfect; rather than building around other brain structures — like the lateral ventricles that transport fluid — the human tissue pushed them out of the way. If that hurt the rats somehow, they didn’t show it. They all performed perfectly normally on a battery of physical and cognitive tests. But it’s the kind of thing that might worry regulators contemplating the first organoid clinical trials.
“At the end of the day, it would be great if we could create layered organoids that could be used for transplantation in stroke patients who are missing parts of their brain,” Waldau said. “And that’s something still left to work toward.”
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