Exploring Autism Through Lab-Grown Brains

The brain of a toddler has more neural connections than that of an adult.

Like caring for a growing plant, these connections are pruned throughout childhood and adolescence, reducing the network to the links, or branches, deemed most important.

By removing redundant or infrequently used connections through a process called synaptic pruning, those remaining become stronger and more efficient.

Inadequate synaptic pruning is among the many mechanisms contributing to autism: a neurodevelopmental difference that shapes how people experience and interact with the world. Reduced or delayed pruning increases connection density, leading to autistic traits such as detail-oriented thinking and contributing to challenges like sensory overload.

Just as the mechanisms behind autism are multifaceted, so is how the condition presents. Some individuals may need daily support and symptom-specific therapies, while others live independently.

To explore how new approach methodologies (NAMs) are contributing to autism research and how insights could inform effective, individualized support, Technology Networks spoke with experts in the field: Dr. Alysson Muotri, professor of Pediatrics and Cellular & Molecular Medicine departments at the University of California San Diego, and Dr. Sergiu P. Pasca, professor of psychiatry and behavioral sciences at Stanford University.

Understanding the autistic brain

Generally, people with autism may face challenges in several areas of daily living: communication, executive functioning, relationships, and sensory processing.

Autism sits on a spectrum; two individuals could experience dramatically different realities. One person may be able to communicate effectively but, under the surface, struggle to navigate social environments. Another person may require substantial support with communication, or communicate in alternative, non-verbal ways.

Pasca recounted his first memory of working with children with autism: “What struck me was both the impact on patients and families, and how little we understood about the underlying biology.”

“Psychiatry was lagging not just because the human nervous system is complex, but also because the relevant tissue, the living human brain, was inaccessible.” — Dr. Sergiu P. Pasca.

Muotri had always been interested in human brain evolution and neurodevelopment, but motivation struck following a personal experience. “I became the father of a beautiful autistic boy. That increased my translational interest,” he expressed.

Recent data estimates that as many as 1 in 31 children live with autism in the United States, a number that is rising as the condition is increasingly understood, recognized, and diagnosed.

While major organizations, such as the National Autistic Society, advocate for autism being seen as a “difference, not a disadvantage,” it is essential to recognize that individuals are at risk of co-occurring conditions that require support and, sometimes, pharmaceutical intervention:

• Individuals with autism face a higher risk of mental health challenges, including anxiety, depression, obsessive-compulsive disorder, bipolar disorder, and schizophrenia.
• They are more likely to experience gastrointestinal symptoms, including acid reflux, pain, diarrhea, and constipation.
• There is a bi-directional relationship between epilepsy and autism, with autism occurring more frequently in individuals with epilepsy and vice versa.
• Learning disabilities occur at a higher rate in individuals with autism than those without.

Given this, researchers endeavor to unravel the complexity of autism and co-occurring conditions to eventually provide personalized treatments that improve overall quality of life and well-being. Autism is increasingly recognized as a unique profile of strengths, rather than deficits, and researchers do not aim to mask differences, but to maximize potential.

A no-brainer: Using brain organoids in autism research

Disease research and therapeutic development have relied on preclinical models. Researchers move from in vitro to in vivo testing; progressing from test tubes, petri dishes, and assays to whole-body systems, i.e., animal models.

Recently, there has been both a push away from this traditional route and a pull toward NAMs—human-relevant modeling systems.

The push has been encouragement toward the 3Rs (replacement, reduction, and refinement), while the pull has been toward systems that better reflect human biology. Although animal models remain necessary for systemic toxicology studies, they cannot accurately replicate human physiology and pathophysiology, leading to failures in translational research.

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Organoids aim to overcome this.

Human brain tissue is a scarce and difficult-to-work-with resource and post-mortem human tissue only captures an endpoint, rather than the process that led to it—this makes it impossible to fully appreciate in vivo disease mechanisms through these samples, leading to the need for an alternative.

Pasca described his journey into brain organoid work: “I came to Stanford to make human neurons from patient-derived cells, initially in two-dimensional cultures, to model disease mechanisms in a human context.”

While the two-dimensional structures represented a breakthrough in the development of human-relevant cerebral models, they lacked the key architecture and complexity required to truly capture the brain's complexity.

“[Our earlier work] set the stage for later development of regionalized three-dimensional neural organoids and then assembloids,” noted Pasca. The earliest organoids most closely resembled the developing fetal cortex, and displayed synapse formation and spontaneous electrophysiological activity.

“Early neural organoids were powerful because they allowed us to model certain features of specific regions of the human brain, but they remained largely isolated systems,” Pasca cautioned.

“The brain does not develop as separate parts. It develops through coordinated interactions between many cell types.” — Dr. Sergiu P. Pasca.

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“Many of the defining properties of the nervous system emerge from complex cell–cell interactions that include synaptic connections, of course, but also other forms of communication among cells within the brain or the periphery,” he added.

This is where assembloids come in, structures that bring together two or more organoids, or fuse an organoid with specialized cell types to investigate cellular and tissue interactions. Pasca pioneered this concept, with his lab being the first to develop three- and four-part neural assembloids.

Muotri highlighted his lab’s contribution to autism spectrum disorder (ASD) specifically: “We were the first group to model ASD using a human system. Compared with animal models, most of the time, the differences are clearer.”

“We found several alterations in brain organoids that we could not detect in animal models, but interestingly, we do see these alterations in postmortem human brain tissues,” he added.

Piecing together autism mechanisms with NAMs

While traditional preclinical models have helped researchers to identify ASD risk genes and the biological pathways they influence, gaps remain.

“Hundreds of genes are now linked to autism and related neurodevelopmental disorders, but we still need to understand which mechanisms are shared and which are specific to individual genetic conditions, and how environmental factors directly contribute,” said Pasca.

Risk genes converge on a handful of processes in ASD. Among these are synaptic pruning, neural connectivity, and communication balances. Organoid and assembloid models are revealing new insights into how disruptions in these processes arise:

• Genetic discoveries: Exploring and identifying autism risk genes and associated cell-type-specific developmental abnormalities.
• Neural circuit function: Investigating the excitatory/inhibitory neurotransmitter imbalance hypothesis, which suggests that hyper-excitable circuits contribute to ASD traits.
• Epigenetics: Identifying DNA methylation signatures that contribute to ASD mechanisms.
• Pharmacovigilance: Determining how environmental factors, such as pharmaceutical exposure, influence neurodevelopment and ASD risk.
• Drug discovery: Evaluating strategies to reverse ASD-associated symptoms.

Pasca highlighted the role of assembloids in identifying morphological features of human cortical interneurons, inhibitory neurons that migrate from the forebrain to the outer layer of the brain during development. The team identified distinct morphological features in Timothy Syndrome, a neurodevelopmental disorder associated with ASD.

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“These examples show how assembloids can help connect disease-associated genes and pathways to specific developmental processes in human cells, while also revealing mechanisms that animal models may not capture.” — Dr. Sergiu P. Pasca.

Autism models guide personalized medicines

The ultimate goal of disease research is to develop accessible treatments that improve patients' lives.

Historically, individuals with ASD have largely relied on supportive therapies such as speech and language and cognitive behavioral therapy to improve communication skills and manage thinking patterns. Pharmacological intervention is limited, with only two medicines approved by the Food and Drug Administration for the management of ASD.

Muotri defined what success in developing new options looks like for him: “Success in autism treatment is related to independence and reduction of clinical symptoms and co-morbidities: seizures, gastrointestinal complications, and inflammation.”

Achieving this may not require a complete mechanistic map of ASD. Instead, Pasca noted that success depends on identifying the key disease drivers and confirming that intervention within these pathways provides real patient benefit.

“Timothy syndrome offers a promising example,” he highlighted. “Starting from patient-derived cells, we were able to uncover aspects of disease biology in human neurons, organoids, and assembloids, and then use those models to begin testing a therapeutic strategy.”

Pasca’s lab developed antisense oligonucleotides that reversed cellular and circuit deficits in Timothy syndrome organoids, including in human neurons in vivo following transplantation into animals. He is currently finalizing preclinical testing for Timothy syndrome mutation, a genetic defect on the cacna1c gene, as he prepares to move his work into clinical trials.

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Researchers are hopeful that the human relevance of patient-derived organoids will improve translational potential. “Unlike animal models, brain organoids allow us to capture the genetic background of the individual in relevant cell types,” noted Muotri.

His group has also seen success, having received Investigational New Drug approval to initiate clinical trials for two different neurological conditions following discoveries enabled by brain organoids. “To me, that marked an inflection point within the field,” he reflected.

Brain organoids provide a unique strategy for ASD research and treatment. Given that they can be developed from patient-derived cells, the end result is representative of patient-specific biology and therapeutic response. Hali et al., used this concept to explore idiopathic ASD—autism with no identifiable cause—in three patients. In addition to identifying neural features shared by all three patients, the team was able to rescue neural circuit impairments using existing drugs, including gabapentin.

Although at an early stage, findings such as these support the continued development of patient-derived organoids as personalized pharmaceutical platforms. This approach allows scientists to move beyond the quest for a universally applicable treatment for a condition that is diverse by definition.

Bridging the convergence of pathways and the divergence of individuals

ASD research has come a long way. The goal has shifted from seeking a cure to curated care, as researchers aim to reduce challenges so that individuality can be harnessed rather than suppressed.

While brain organoids have allowed researchers to take important steps toward this goal, questions remain.

Pasca highlighted the importance of understanding the interplay between genetics and environment: “A central question is how convergence and divergence emerge across the many genetic forms of autism. This distinction matters for both biology and therapeutics, because convergent pathways may point to broader interventions, while divergent mechanisms may require precision approaches.”

“A second major question is timing,” he added. “We need to know which developmental processes are altered, how early those changes arise, and whether they can still be corrected later. The question is: ‘How early is early enough for an intervention, and when is it too late?”

But unanswered questions will remain if challenges are not addressed. Muotri highlighted the need to improve the translational potential of brain organoids and to address costs with the modern models to facilitate ongoing research.

“If we improve the quality of life and independence of autistic individuals, we are on the right track,” he concluded.