‘Brains Within Brains’: Organoid Experiments Show How Pathologies Emerge in the Developing Brain
Pioneering grantees have harnessed stem-cell technology to grow unlimited numbers of human brain cells in the laboratory. In their most recent innovation, they have transplanted assemblies of these cells, called “organoids,” into living animal brains, where they make connections and begin to function. This is making possible unprecedented experiments to reveal pathologies in human brain illnesses, particularly those like schizophrenia and autism with developmental origins, and provides a unique test-bed for assessing new therapeutics.
Sergiu P. Pasca, M.D.
Kenneth T. Norris, Jr. Professor of Psychiatry and Behavioral Sciences
Bonnie Uytengsu and Family Director of the Stanford Brain Organogenesis Program
2017 BBRF Independent Investigator
2012 Young Investigator
Those who study illnesses rooted in the early brain like schizophrenia and autism spectrum disorders face an obstacle that most medical researchers don’t. While they have ample access to patients, they have no access at all to living, functioning tissue of the organ in which pathology is presumed to be centered.
2017 BBRF Independent Investigator and 2012 Young Investigator Sergiu P. Pasca, M.D., of Stanford University, explains the situation that prevailed at the very beginning of his career, in the early 2000s: “I’m a physician by training, and my interest has always been in understanding the biology of neuropsychiatric disorders. I found it very frustrating to try to do this research without having access to brain tissue from patients.”
Dr. Pasca is well known as a key innovator of a technology that addresses this seemingly insoluble problem. The solution, as this story will explain, builds upon powerful insights about stem cells, sometimes referred to as “the mothers of all cells.” Dr. Pasca and other investigators have figured out ways to grow unlimited numbers of human brain cells in the laboratory, and, in their most recent innovation, to transplant assemblies of these cells, called “organoids,” into the brains of living animals, where they make connections and begin to function. These transplanted organoids become, in effect, brains within brains—segments of the human brain living within a fully functional animal brain. This is making possible unprecedented experiments to reveal pathologies in human brain illnesses, particularly those with developmental origins, and provides a unique test-bed for assessing new therapeutics.
In the past there have been many meaningful efforts to cope with the problem of access to the living human brain. Collections of postmortem human brains have been assembled and archived for research, thanks to the great generosity of families whose loved ones lived and died with mental illness. These collections have supported many important studies, but such research can by definition only go so far. Some of the central questions of biological research—showing how complex living systems function in real time and how they change over time—need to be explored in living, functioning brains.
Brain scans and other non-invasive technologies like EEGs (electroencephalograms) that reveal function in the living human brain have also been powerful tools for researchers. But these technologies also have their limits—as do animal models of human disorders. It’s possible to observe behaviors in animals that resemble those seen in some human psychiatric illnesses. But again, there are limits: no mouse or rat can ever be said to have schizophrenia, bipolar disorder, or autism. These are uniquely human disorders, defined by changes in human perception and behavior.
TURNING TIME BACKWARD
In 2006, a seminal discovery was made by Dr. Shinya Yamanaka, of Japan, which brought him the Nobel Prize six years later. Dr. Yamanaka was interested in stem cells, particularly pluripotent stem cells, which at the beginning of life populate the embryo. These precursors are capable of developing into all of the cell types that make up the adult organism. As the weeks pass, stem cells give rise to specialized cells that form the organs of the body. Before Yamanaka, this journey from immature to specialized cell was assumed to be unidirectional—once specialization occurred, there was no way for a cell to return to an early, pluripotent stage.
Through trial and error, Yamanaka identified a set of just a few genes whose activation in cells effectively acted like a time machine—the cells turned back into pluripotent stem- like cells. This worked first in mouse cells, but soon was shown to be just as effective in human cells.
It was now possible, in other words, to sample mature cells from an organism—humans included—and return them to a pluripotent state. In the lab, these pluripotent cells, grown in culture dishes, could then be induced to re-develop as any of a variety of specialized cell types. Something as innocuous as a skin cell, which can be sampled painlessly from any individual, could be returned to a stem-cell-like state in the lab, and then reprogrammed to redevelop as, 16 Brain & Behavior Magazine | September 2023 say, a neuron. There was now a path to generating an unlimited supply of living brain tissue.
This game-changing technology made possible research that Dr. Pasca and so many other neuroscientists wanted to perform. It had an imposing name: “induced pluripotent stem cell” technology, or iPS. By the time Dr. Pasca was awarded a Young Investigator grant by BBRF in 2012, he had already developed some of the first models with iPS cells by generating neurons in a dish from patients with a form of autism caused by a genetic mutation.
It took some time to make good on the promise of iPS technology. “We were able to make these beautiful cultures at the bottom of a dish, cultures of neurons,” Dr. Pasca remembers. “And we found that we could easily keep them for 10 weeks or so, if we fed them.” That was exciting. “But we found that this could only go so far. We could not, with this set up, really recapitulate later, key stages of brain development.” In the emerging human fetal brain, for example, it takes more than 20 weeks to generate all the neuronal types found in the human cerebral cortex— something that iPS technology in its early version was not able to sustain.
Dr. Pasca had an idea. His cortical neurons generated with stem cell technology were laying at the bottom of the culture dish; why not try to grow them so that they were suspended in three-dimensional space? A special coating applied to culture plates made the cells lift off the surface and float. These neurons were more dynamic. They formed balls of cells that self-assembled and could be kept alive indefinitely. Each ball started with about 10,000 cells but could grow to contain several million. “We’ve maintained them for as long as 800 or 900 days,” Dr. Pasca says.
ORGANOIDS DERIVED FROM PATIENTS’ CELLS
In a series of papers, Dr. Pasca and colleagues showed that over long periods of time, these balls of brain cells, called organoids, “will develop at pretty much the same pace as they would in the living context.” Remarkably, “after nine months of keeping them in a dish, they transitioned to a postnatal signature. This transition in signature from fetal to postnatal occurs at about 280 days and tells us there’s an intrinsic clock, a maturation clock, built into these cells.”
What made these early organoids potentially so powerful was the fact that they could be generated from skin cells harmlessly sampled from any person—including people with psychiatric (or other) illnesses. Dr. Pasca and others were especially eager to create organoids derived from cells sampled from patients with disorders like schizophrenia and autism thought to have roots in early development, when the fetal brain is just emerging.
In organoids based on cells sampled from patients, every cell has the genome of the patient-donor. If this donor has genetic mutations linked with high risk for disease pathology, then a novel kind of experiment becomes possible. One can watch these cells from their earliest days as they develop and begin to manifest pathologies caused (at least in part) by their risk-related variant genes.
In an important paper in Nature Medicine in 2020, Dr. Pasca and colleagues provided a vivid example of how stem cell-based technology could help reveal pathological mechanisms in neurodevelopmental illnesses. The subject was an illness called 22q11.2 deletion syndrome, which is associated with schizophrenia and autism spectrum disorder. It’s caused by a chromosomal deletion of about 60 genes.
His team generated organoids composed of cells reprogrammed to redevelop as cortical neurons—the cells that populate the brain’s cerebral cortex. In organoids derived from over a dozen patients, neurons showed deficits in how they “fire,” as well as in how they handle ions of calcium, which help regulate voltage in cells. This was evidence of at least one of the pathologies in 22q11.2 deletion syndrome that likely relates to its devastating impact on patients.
FROM LAB DISH TO LIVING BRAIN
Dr. Pasca’s early brain organoids showed signs, albeit partial, that they marked the transition from prenatal to postnatal. But, he notes, “these cells in the organoid were modeling just one brain region—the cortex.” To model multi-region brain complexity “we built the first assembloids.”
Assembloids are combinations of three-dimensional organoid cultures that represent different regions of the brain. Cells of the cortex, including neurons and helper cells called astrocytes, formed organoids that were combined with organoids composed of cells found in the striatum, or, in other experiments, the spinal cord. “We started putting more brain regions together and looking at the connections between them.”
There was much more to do. “Even with these models, and even being able to maintain these cultures for hundreds of days, we realized that there were still some properties of the cells that we were not capturing in the dish. For instance, neurons still do not grow in culture as large as they are in the actual human brain. They do not become fully mature.”
There were also questions about their functional properties. “If we wanted to understand the biology of psychiatric disorders, we had to find a way to enable human neurons to influence circuits in the context of the living brain,” Dr. Pasca says. “That’s why, about 8 years ago, we started playing with the idea of transplantation—the possibility of transplanting intact three-dimensional cell cultures directly into the rodent brain.”
In 2018, a team led by BBRF Scientific Council member and 2013 Distinguished Investigator Fred “Rusty” Gage, Ph.D., of the Salk Institute for Biological Studies—for decades, a pioneer in stem cell-related technologies to study the brain—published a paper in Nature Biotechnology introducing a method of transplanting human brain organoids into the adult mouse brain. These ”grafts” were observed to generate a variety of cell types which matured in the rodent brain environment. Remarkably, the team observed connectivity develop between the human brain-cell graft and the rodent brain host, and the formation of synapses between neurons in each that appeared to generate connectivity that could affect the animals’ activity.
In 2022, Dr. Pasca and colleagues reported in Nature on experiments in which they grafted intact human-derived cortical organoids into the brains of rats that had just been born. The hope was that the organoids “would actually grow and become a unit within the rat’s cortex, and in a very specific position.” The targeted location was the rat’s somatosensory cortex, which was easy to access. It was also targeted because this part of the cortex receives abundant input from the thalamus, a kind of relay station, where inputs arrive from the rat’s whiskers, the animal’s primary source of sensory information.
“We did the transplants in the first week after the rats were born, when the animal’s brain circuits are still forming.” The results were “remarkable,” Dr. Pasca says. Within 8 months, the transplanted organoids grew to nine times their pre- transplantation volume and, as revealed by MRI, came to occupy about one-third of a hemisphere of the rat brain. Not only were the transplanted neurons larger; they also formed more complex branching connections with other brain cells than did neurons grown in the lab. The rodent hosts receiving the organoid transplants steadily supplied the human neurons with nutrients and electrical inputs, a measure of their successful integration.
This was no stunt. The team went on to conduct experiments demonstrating that the human neurons within the rat brain began to respond to inputs the rats were receiving from their whiskers. In other words, the human cells were integrating functionally and could receive sensory stimulation. And in what might be their most consequential success, the team engrafted cortical organoids derived from cells donated by patients with Timothy Syndrome, a disorder that shares many clinical features with autism spectrum disorders. These organoids developed and integrated with the host brain in ways that clearly revealed pathologies consistent with the illness.
Beyond making it possible to observe the origins of pathology, the technology creates new opportunities to test potential therapeutics for developmental disorders “Very often,” Dr. Pasca says, “animal models don’t recapitulate them well enough to gauge the impact of a newly developed drug.” But testing candidate drugs in animals with highly integrated patient-derived organoids might be particularly helpful in determining a drug’s impact on the pathologies that emerge in the organoids. In the last year, Dr. Pasca has created a new team within his lab dedicated to developing therapeutics, including one, he says, that appears to have promise in a neurodevelopmental disorder with genetic roots.
FACTORING IN THE IMMUNE SYSTEM
Among the questions that define any set of experiments with brain organoids is “what types of cells do you want to have in the organoid?” This observation, by Simon T. Schäfer, Ph.D., one of Dr. Gage’s mentees at Salk who has established his own lab at the Technical University in Munich, Germany, establishes a context for another important milestone in organoid-based experiments.
In a recent paper appearing in Cell, Dr. Schäfer, a 2021 and 2018 BBRF Young Investigator, along with Dr. Gage and other colleagues, reported success in engrafting into the rodent brain an organoid consisting primarily of human cortical neurons, but importantly, also including an important cell type called microglia. Microglia are the only cells of the body’s innate immune system that live and function in the human brain. In the healthy brain, these cells are constantly surveilling the environment, looking for toxins and responding to damage. No one had previously succeeded in growing a human brain organoid with microglia that completed the journey to functional maturity.
Microglia are generated from a fundamentally different stem cell precursor type than other cells of the brain, such as neurons, helper cells like glia, or fatty cells called oligodendrocytes which protectively insulate nerve pathways. Those and other cells of the human nervous system are products of stem cells from one of the three layers of the early embryo called the ectoderm. Microglia derive from stem cells in the original embryo’s mesoderm, which is also the source of all blood cells.
Microglia can be generated in the lab and added to organoids composed of neurons. But they fail to thrive. Drs. Schäfer, Gage and colleagues wanted to see what would happen if microglia were incorporated in the lab into a human-derived cortical organoid and then immediately transplanted into the living rodent brain.
Their hypothesis was that signals sent and received only in the environment of a living brain might enable the microglia to mature and start to perform their immune surveillance function. This is precisely what happened. Once a protective layer called the blood-brain barrier forms, microglia become “trapped” in the engrafted organoid, just as they do in the developing human brain.
The microglia then began to respond and function when they sensed factors emanating from the human cells within then engrafted organoid. Now it was time to perform a parallel experiment. Just as Dr. Pasca’s team had implanted into the rodent brain cortical organoids derived from patients with Timothy’s Syndrome, Dr. Schäfer and colleagues now grew cortical organoids “colonized” with primitive microglia derived from patients diagnosed with autism and a co-occurring condition called macrocephaly (enlarged head size, which in severe cases has serious neurological and developmental consequences).
Two remarkable observations followed. One was that inside these patient-derived organoids functioning within the rodent brain, the microglia became very active. The team thinks this recapitulates something that happens in the brains of children with the combined condition.
The other observation was that intense microglial activity resulted in inflammation within the engrafted organoid. And this is important because brain inflammation is often seen not only in autism, but a host of other neuropsychiatric conditions including schizophrenia and depression. Itself a source of pathology, inflammation has been hard to study in patients, but the organoid transplantation strategy provides one way to do just this.
The team’s observations led to a crucial question: why did microglia become so active in these models of patient brain tissue “living” within the functioning rodent brain? Was there something about the microglia themselves that accounted for their unusual activity? Or was their activity dependent upon something in the organoid environment in which the microglia were functioning? Sophisticated experiments were performed which led the team to conclude that it was not the microglia, but signals from their immediate environment—that unique environment of a living, functioning brain—that prompted their high level of activity, leading to the inflammation that likely has a role in the pathology of the combined condition of ASD + macrocephaly.
“We knew from autopsy and biopsy tissue that patients with autism often show inflammation,” Dr. Schäfer explains. “Well, we wanted to know where this inflammation comes from. The experiments suggested it is the developing brain environment that changes the activity of the microglia, which may result in inflammation.”
This is something that begins early in development, it appears, and, says Dr. Schäfer, “it probably has very long-lasting consequences” for patients. “I think this also may prove relevant in other disorders, where you see contributions [to pathology] that are immune cell-driven.” A new question the team now studies is whether microglia, in the very early stages of brain development, are, in effect, “trained incorrectly” due to environmental signals, such that they become overactive, opening the way to inflammation. The experimental approach is to perform experiments with transplanted organoids that enable the team to observe these processes as they occur, ideally when they first occur.
From such insights, it is hoped, may come new concepts for therapeutics. “We’re just now building a center for organoid systems,” Dr. Schäfer says. “We’re bringing together people with different kinds of expertise to build systems that we can translate, not only to disease models, but maybe also to treatments.
“This is why I’m interested in organoids. We’re used to doing science in a certain way: we make deductions based on our observations of complex systems [in which pathology is already present]. With organoids we have the chance to observe things from the very beginning of the process.”
Written By Peter Tarr, Ph.D.