“Are you ready to make some organoids?” asks senior research scientist Se-Jin Yoon. Two dozen PhD students and postdocs from labs around the world whisper excitedly. Clad in white lab coats, jeans, and sneakers, they’ve assembled at the Wu Tsai Neurosciences Institute on campus to participate in one of Sergiu Pașca’s workshops. Today, they’ll cultivate pea-size balls of human neurons that will help them in their research on various neurodevelopmental disorders, from schizophrenia to autism spectrum disorder.
Pașca, a professor of psychiatry and behavioral sciences at Stanford, is a trailblazer in the field of stem cell brain modeling, also sometimes known as molecular psychiatry. For 15 years, he’s made increasingly complex living tissue models of the human brain—a “black box” in medicine. It is estimated that one in four people has a neurological or psychiatric disorder. Studies have implicated hundreds of genes in these conditions, yet we know little about how to treat them. “We are farther behind than in any other branch of medicine,” Pașca says. Cumulatively, these disorders are “enormously impactful on the patients, their families, and communities,” says Karl Deisseroth, PhD ’98, MD ’00, a professor of bioengineering and of psychiatry and behavioral sciences at Stanford and one of Pașca’s collaborators. “All are challenged by our lack of understanding of what is fundamentally going on” in the brain.
One of the major obstacles to research into neurological and psychiatric conditions is the fact that scientists can’t take brain samples from living people. Pașca’s models of human tissue allow scientists to noninvasively examine the development, function, and dysfunction of the human brain in real time, and, crucially, with neurons that actually fire. The results have Pașca on the cusp of a treatment for a rare and serious genetic disorder called Timothy syndrome, which counts autism among its most common symptoms. And, he says, addressing other neuropsychiatric conditions may not be far behind.
Good chemistry
Pașca exudes energy. At 42, he’s a brisk-walking, hard-driving machine. The child of a schoolteacher and a plumber who later built a construction business, he grew up in a small apartment in Transylvania, a region in central Romania. As a 5-year-old, Pașca liked school and looked forward to Sunday mornings, when he would watch his favorite show on the family’s black-and-white TV. It included a segment with dictator Nicolae Ceaușescu’s wife, Elena, who, despite dubious credentials, was called a world-renowned scientist and talked with Romanian scientists about their experiments. “In general, in communist countries, science was held quite in esteem,” Pașca says. “That was the way you could contribute and improve society.” The Ceaușescus were executed in the 1989 Romanian Revolution, but Elena’s science segments left an imprint on Pașca. He went on to win his high school’s chemistry Olympiad, which awarded him admission to his choice of college, and in 2001, he chose Romania’s Iuliu Haţieganu University of Medicine and Pharmacy. There, he thought, he could learn to treat patients and start a career as a lab researcher—like those scientists on Sunday morning TV. But when he finally walked into a laboratory at the university, the state of biomedical research in Romania shocked him. “I realized most of the reagents, most of the machinery needed for research, was from the ’80s,” he said. Based on what he had read about lab discoveries in other parts of the world, he realized his path to becoming a world-class lab scientist wouldn’t be easy.
He started to look for an area of medicine in which relatively little was known—where he might make discoveries using old and limited tools. He was drawn to his patients with autism, a condition then so poorly understood that he believed small studies could reveal new insights. “At least now we know hundreds of genes are associated with [autism],” he says. “At that time, we didn’t know anything.” He started with blood samples from his patients, trying to identify anything that would tell him about the biological basis of neurodevelopmental disorders. When he told one mother he was working to understand autism, she burst into tears. “She embraced me. I’ve never had a reaction like that. I wanted to help these families.” (Autism, it should be noted, exists along a spectrum, and many with the diagnosis celebrate its strengths; not everyone experiences distress or wants to be cured.)
While in medical school, Pașca published several small studies uncovering abnormal blood biochemistry in patients with autism, including elevated levels of homocysteine, which also has been associated with neuropsychiatric conditions such as major depressive disorder and schizophrenia. But he soon realized studying blood wouldn’t be enough. Whatever was happening in the brains of people with neurodevelopmental disorders was happening, well, in the brain. “The blood is so far away from electrical activity,” Pașca says. He decided to pivot to neuroscience. And since there were limited lab supplies and effectively no neuroscience in Romania at the time, “I decided to leave.”
‘She embraced me. I’ve never had a reaction like that. I wanted to help these families.’
After graduating from medical school in 2007, Pașca reached out to Ricardo Dolmetsch, PhD ’97, asking for a post-doctoral position in his Stanford lab. Dolmetsch, then an assistant professor of neurobiology, had studied the basic biology of nerve cells. But in 2006, his son was diagnosed with autism, and Dolmetsch shifted his research focus accordingly.
“I’d heard he was interested in autism, and I’d published on autism,” Pașca says. But Pașca had never done cell culture work in a lab. He’d only started learning English a few years before. And he didn’t have a PhD. “I was coming from nowhere.” Still, Dolmetsch replied that he might have a spot in his lab—if Pașca could secure his own funding. Thus began more than a year of work applying for grants. “At one point, I asked Ricardo, ‘Why did you even want me?’” Pașca was persistent and ambitious—and had potential, Dolmetsch told him. That was more important than his existing knowledge. Pașca finally landed a fellowship from the International Brain Research Organization in 2009. “When I came here, I kind of felt like a fraud,” he says. “I’d never done experiments with cells in my life, so, essentially, I worked all the time to try to catch up.”
But Pașca arrived at Stanford with a plan that had been incubating since he’d graduated from medical school. That year, he’d read about a Japanese researcher who’d generated induced pluripotent stem (iPS) cells—the kind that can become any type of cell in the body—from skin biopsies, bypassing ethical questions circling at the time around the use of stem cells from human embryos in research. Using this method, Pașca believed he could chemically coax such stem cells into becoming brain cells to make some of the first lab-grown neurons—observing their development from the very beginning.
One small change
The Dolmetsch lab had been studying Timothy syndrome, an exceptionally rare and often life-threatening genetic disorder that is thought to affect about 70 people worldwide. Children with the disorder rarely survive to late adolescence. It is caused by a defective gene on the 12th chromosome that codes for a protein with a pore that controls the flow of calcium across cells’ membranes. This calcium channel controls many of the processes a cell needs to function. In Timothy syndrome, the calcium channel is overactive, staying open for longer periods and allowing more calcium than it should into the cells. This has widespread consequences, including deadly problems with the heart’s electrical system (these days often controlled with a pacemaker), epilepsy, developmental delays, severe psychiatric symptoms, and an 80 percent chance of developing autism.
Pașca and Dolmetsch believed that if they could understand how a calcium channel managed to cause the neurological effects of Timothy syndrome, what they learned might also apply to the root causes of other neurological conditions. “Rare syndromes that are very clearly defined genetically are sort of like windows, or Rosetta Stones, into understanding other, more common conditions,” Pașca told NPR last spring. Now he just needed Timothy syndrome neurons.
Within six months of arriving at Stanford, he had them. Pașca turned the skin cells from a young patient with Timothy syndrome into iPS cells, and then into neurons. Other scientists were skeptical that the disease would show itself in these lab-grown neurons. By erasing the mature identity of the cell, they said, he’d probably erase the disorder as well. But in a 2011 publication, Pașca showed that the lab-grown neurons had the same cellular defects associated with the disease. “It was unbelievable,” he says. “You could do the calcium imaging and you could see that there was a difference.”
Now, the Dolmetsch lab had a model for Timothy syndrome—a way to study how the genetic disease altered brain development, and possibly explore treatments. “People thought it was very far out,” said Dolmetsch in a 2011 interview with the Transmitter. “But we’re now convinced this is a very viable approach to studying autism.” The study made the cover of Nature Medicine. “That got a lot of attention, and got me a green card too,” Pașca says.
Brain cells, assemble!
By 2014, Pașca was an assistant professor of psychiatry at Stanford and had established his own lab. He’d had Timothy syndrome patient neurons for a few years, and he’d been recruiting patients with other disorders. His plan was to study early brain development via his lab neurons, watching as they grew to see where the disease-model neurons varied from the controls. But the neurons didn’t survive long enough. “They couldn’t live past 100 days,” Pașca says.
Human brain development is simultaneously mind-blowingly fast and a little like watching grass grow. On the one hand, a fetal brain generates about 3.86 million neurons per hour. On the other, the neurons need to differentiate into multiple types, and that takes months. “I realized we needed the neurons to get past at least 20 weeks for the different types of neurons to be born,” Pașca says. He surmised that the neurons were dying off because they were sticking to the petri dish, so he used dishes with a nonstick coating. The freed 2D neurons began to self-organize into floating 3D spheres of thousands of neurons. (A human brain has close to 100 billion neurons.) The lab had made some of the first-ever brain organoids. The phone started ringing. Other labs were on the line, asking Pașca to share his methods, which he did happily. Some in the media, on the other hand, dubbed the balls of cells mini-brains. “It wasn’t all great,” Pașca says. “I got called, like, a witch or a wizard brewing brews in a dish. I was referred to as ‘Frankenbrain’—you know, ‘that scientist from Transylvania.’”
That didn’t stop him. “We want to find answers to solve disease,” Pașca says. He was convinced he could keep the neurons alive long enough to provide those answers. By adjusting the nutrients and growth factors in their recipe, his team began to create different types of organoids, each one mimicking the architecture and physiology of a single region of the brain—for example, the cerebral cortex, the outermost layer of nerve tissue in the brain. Today, Pașca’s lab has some of the oldest living organoids, which have thrived for more than 1,000 days in a dish. “In theory, we could keep them forever,” Pașca told the students in his workshop.
MODEL MINDS: Paşca’s lab is dedicated to finding the molecular mechanisms that lead to neuropsychiatric disorders.
Even so, looking at organoids corresponding to single regions of the brain wasn’t giving Pașca the full picture. He knew Timothy syndrome neurons were atypical: The defective calcium channel hindered their growth. But he still needed to find out how that resulted in atypical development for the brain as a whole. Perhaps, he thought, the neurological effects of the disease stemmed from a faulty interaction between brain regions. So he put an organoid mimicking a brain region called the subpallium into glassware with an organoid mimicking the cerebral cortex. Early in the development of a human brain, the subpallium extends migrating neurons to connect with the cerebral cortex. Pașca discovered that when placed together in a lab dish, the organoids similarly fused and self-organized, mimicking the complex neuronal connections that occur during fetal and neonatal development. “The human brain largely builds itself,” he says. He dubbed these new models assembloids.
Now, Pașca and his team could examine how migrating neurons—called interneurons—moved from one region to another. In the nondisease control models, they did so in stuttering jumps, and they formed vast trees with branching dendrites—antennae for receiving signals. In a 2017 Nature publication, Pașca’s team showed that in the assembloid models with Timothy syndrome, the interneurons’ jumps were much smaller, their antennae withered. “And then there is this migration of these interneurons that are not arriving at the right time” in development, Pașca explained on the Stanford podcast From Our Neurons to Yours, produced by the Wu Tsai Neurosciences Institute. “They’re probably arriving with a delay.”
The big questions
Even assembloids have their limits. Without the input of an entire brain, they don’t grow as large or have as much electrical activity as they would in a whole, living brain, says Deisseroth. “You really need blood vessels as well as [widespread] neural circuitry.”
In a 2022 Nature paper, Pașca and his colleagues described a way to provide his lab-dish neurons with that whole-brain environment. It involved the transplantation of lab-created human cortical organoids into the brains of young rats and resulted in the formation of working, hybridized brain circuits. When a rat’s whiskers were tickled, the human neurons responded, helping generate signals to guide neuron development. The organoids integrated into the rat’s brain without prompting, grew to six times the size they were in the dish, and developed more complex brain patterns. This living lab allowed Pașca to observe differences in the behavior and development of the Timothy syndrome neurons that couldn’t be seen in petri dishes. It also, perhaps unsurprisingly, raised eyebrows. Within days of the study’s publication in Nature, more than 2,000 media stories sprung up, some raising questions about the ethical implications of such hybridizations.
The use of rodents in research is widespread and generally accepted, says bioethicist Insoo Hyun, ’92, MA ’93, director of the Center for Life Sciences and Public Learning at the Museum of Science in Boston and an affiliate of the Center for Bioethics at Harvard Medical School. But to many people, transplantation of human brain organoids into a rodent’s brain felt different. “People believe [the brain is] the seat of our experiences, our memories. That’s where you get all the controversy,” Hyun says.
Hank Greely, ’74, a professor of law and the director of the Stanford Program in Neuroscience and Society, points out that human and nonhuman cells have been used together for decades—for example, by replacing damaged human heart valves with valves from pigs. The concern here is with brain research, he says. There could conceivably come a time when an animal-human brain model might begin to show human-like characteristics or consciousness. But “we don’t have any models that are that good yet” and may never, says Greely, who served on a U.S. National Academies of Sciences, Engineering and Medicine panel that concluded in 2021 that human brain organoids were still too primitive to become conscious or attain any humanlike characteristics.
Hyun is not worried about these animal models developing humanlike traits. A transplanted organoid contains about 3 million neurons—about 1/67th of a rat brain. But he does think growing brain organoids in animals more complex than rats (say, primates) would raise questions about the potential of humanizing them. “I don’t think there is any kind of justification for this [type of animal study],” he says, nor does he know of any labs attempting such research.
IT TAKES TWO: An assembloid formed by the joining of two organoids.
Pașca, who has helped organize scientific committees to consider the ethical implications and potential regulations surrounding his field, says his lab takes care to test for any potential humanizing of the rats. “We did a battery of cognitive tests and saw no difference in memory, fear conditioning, and other conventional tasks in rats,” he says. That is: They remained rodents. But he understands the concerns and holds some himself. “As our models are getting more elaborate, and with building human circuits in animals, there are a number of ethical issues to consider,” he says, “including the welfare of the animal and any possibility of emergent [human] features.”
Hyun says regulation and oversight of these models should continue to evolve, and bioethicists should be actively working with labs to help steer where things go from here. Right now, he believes, the ethical focus should be on improving the consent process for donors whose cell lines are used in this field. A patient may consent to have their skin sample taken when it’s destined for a petri dish. Neither they—nor the researchers—necessarily know at the time what future research the cells could be used for. “Do you ever have to go back to donors and make sure it’s aligned with their values?” he says. “Because this issue is so ethically sensitive and culturally important.”
Greely agrees that oversight and regulation is needed—and emphasizes that so, too, is this research. “Brain disease is one of the biggest sources of human suffering in the world and we’re almost nowhere with treatments,” he says. “We have a strong moral imperative to do this type of research.”
In fact, the rat model proved to be the final link in the chain toward discovering the Pașca lab’s first potential therapy. Pașca and his team published a 2024 study in Nature demonstrating the ability of antisense oligonucleotides (ASOs) to bypass the defective calcium channel that results in Timothy syndrome. An ASO is a piece of a nucleic acid that can change gene expression. When administered into neurons with the Timothy syndrome mutation, they prompt the neurons to use an alternative, nondefective calcium channel. Within days, Pașca showed, the neurons had started to use the alternate calcium channel and cellular defects were reversed.
In other words, the neurons that initially had Timothy syndrome developed as if they didn’t have it—using the alternate calcium channel, making bigger jumps to other parts of the brain, sprouting long branches. Pașca hopes to start a human clinical trial early next year. “It’s the culmination of all these years of work,” Pașca says. “It’s not that Timothy syndrome was difficult to understand mechanistically. We just needed the models.” If the treatment is able to mitigate some of the neurological effects in kids with Timothy syndrome, the same approach could eventually lead to treatments for other conditions, including schizophrenia and bipolar disorder, Pașca says. He believes the Timothy syndrome treatment may essentially “unlock” treatments for numerous neuropsychiatric disorders.
Picture it
Pașca leans back in his office chair. He’s got the blinds closed and the lights low so he can concentrate on work. Outside his office door, lab workers are busy at their microscopes. He’s recovering from both jet lag and a cold, having recently returned from a work trip to Japan.
In the dim light, a wall of bright colors catches the eye. It’s a litany of framed covers of science journals—a showcase of his work. He takes down a 2024 Nature cover, the one that includes his paper proposing the potential therapy for Timothy syndrome. The cover image is a photograph shot by his high school best friend, now a photographer in Romania.
BRAIN SPACE: Pașca aims to deliver cures.
“It’s a lake [photographed] from a drone in Romania when the ice cracks in winter,” he says. “When I saw this, I was like, those are the shape of neurons.” He points to the bigger, star-shaped cracks. Those symbolize the control neurons with long dendrites extending out to communicate with other neurons. Next to them are much smaller cracks, the neurons that couldn’t sprout and branch as far.
“I made myself a promise 15 years ago,” Pașca says. “After seeing one of my first patients with autism, I realized that as a physician, I could only see one patient at a time. I wanted to deliver cures.” If he couldn’t find any promising therapy leads as a researcher, he would return to medicine. That seems increasingly unlikely. “My plan now is to make therapies that help patients.”
Tracie White is a senior writer at Stanford. Email her at traciew@stanford.edu.
