Human Cerebral Organoids Transplanted Into Newborn Rats Integrate With Their Brain

A new study has advanced our ability to model the developing brain by implanting balls of cells – called brain organoids – grown from human stem cells into the brains of young rats. The organoids, which are usually grown in a dish, showed unprecedented levels of maturation, formed connections with other areas of the rat brain and were even capable of stimulating changes in the animals’ behavior.

The research marks a step forward for organoid technology, which has become a valuable tool in developmental neuroscience, but raises pressing ethical questions about animal welfare and the level of consciousness that more complex organoids could one day obtain.

The research was published in Nature.

The origin of organoids

In 2006, Kazutoshi Takahashi and Shinya Yamanaka published a seminal paper that showed cells could be reprogrammed to an embryonic-like state, where they are capable of being specialized into varied other cell types through a process called differentiation, with the application of a handful of genetic drivers called transcription factors.

Since then, science’s ability to model the developing of cells using stem cells has rapidly advanced. Researchers at Stanford University led by Sergiu Pasca, a professor of psychiatry and behavioral sciences, have been a central force in advancing neural stem cell models. Pasca and colleagues published influential papers showing how neurons derived from stem cells could be used to model disorders, such as the rare genetic condition Timothy syndrome, which produces malformations of the heart, nervous system and digits.

But early stem cell models, which were arranged in monolayers along the bottom of dishes, lacked the structural complexity and circuitry of the brain. Researchers around the world began the drive towards developing 3D stem cell models – organoids – that more faithfully modeled neural processes. But while organoids in dishes proved to be useful models of the early brain, their growth tended to stall at certain developmental timepoints: with no blood running through them and lacking support from a wider nervous system, these organoids remained small and relatively simple compared to the mazy connection maps drawn by the mature mammalian brain.

Pasca’s lab, with their chimera mice, may have cracked the problem.

An unprecedented level of integration

With in vitro brain organoids, says Pasca, “we can’t really tell what are the behavioral consequences of defects that we identify.” This has limited their use in studying disorders like Timothy syndrome, which produce complex intellectual and behavioral changes.

To try and bridge the gap between the behavior produced by a brain and the genetic changes shown by brain organoids, Pasca and his team transplanted whole organoids, roughly 1–1.5 mm in size, into the developing brains of young rats. While transplantation studies are hardly new, he says, his team’s study had two innovative features – the complexity of the organoids and the timepoint (3–7 days after birth) at which they were added. “By transplanting them at these early stages,” says Pasca, “we found that these organoids can grow relatively large, they become vascularized and they grow to cover about a third of a rat brain hemisphere.”

Pasca’s team added their organoids to a region of the rat brain called the somatosensory cortex, which receives input from the rat’s whiskers. The level of integration that the organoids achieved was unprecedented, taking root in 81% of the 72 rats transplanted. The level of connection the organoids achieved was so intimate that the human-derived neurons even responded to stimulation of the rats’ whiskers. Support cells called microglia produced by the rat even wound their way into the grafted cells. The rats used lacked a thymus, meaning their immune systems were defective, stopping their bodies from rejecting the grafted cells.

Once in place, the neurons also showed physical changes that much better mimicked in vivo brains, growing to around six times the size of neurons grown in a dish and showing more complex electrical activity. The transplants differentiated into a range of cell types, but failed to show the layered arrangement, called lamination, that normal rat cortexes form. The organoids had far more complex arrangements of dendritic connections and were capable of stronger electrical firing rates than equivalent dish-grown organoids. After roughly 8 months of growth, analysis of the organoids’ transcripts showed that these gene products were similar to those produced in the late fetal period in intact human brains.

Modeling disease

The team then transplanted organoids grown from the stem cells of patients with Timothy syndrome. While organoids from these patients grown in a dish looked similar to those created from individuals without Timothy syndrome, the transplanted organoids grew abnormal dendrite patterns, suggesting that some insights into neurodevelopmental disease might only be attained using the novel system.  There were also changes to how much Timothy syndrome-patient derived cells grew after transplantation, “Patient cells don’t grow as large. There’s a difference that you can literally see by eye,” explained Pasca.

Perhaps the most striking feature of the transplanted organoids, however, was the extent to which they networked with the rats’ existing brains. The team used a host of genetic tagging techniques to map and modulate the transplants. They showed that not only did the transplants receive inputs from the rest of the rat brain, but that light-based stimulation targeted at the implants were able to change how the rats performed in behavioral tasks.

Assessing the risks of organoids

Madeline Lancaster, a scientist at the MRC Laboratory of Molecular Biology, who pioneered some of the first organoid research and was not involved in study, called the work a “step forward” for the field. “[The study] offers a new way to understand disorders of neuronal functioning using a human model system,” said Lancaster. Pasca suggested that the system could be used as a new kind of drug platform, sitting in between in vitro pre-clinical work and human in vivo clinical trials.

While stem cells have produced their own moral panics, the risks of dish-grown organoids attaining sentience or consciousness have been minuscule. But as the technology moves into biological systems and gains in complexity, how have those risks changed? Pasca explains that his team worked closely with internal and external bioethicists throughout the study, with a priority on animal wellbeing. “One of the main concerns that we’ve had is that they would have seizures or epilepsy,” he explains. But electrical and physiological analysis suggested that the rats had no increased risk of seizures or increases in stress or deficits in memory. “To the extent that we can tell, there are no alterations to the rats’ behavior or wellbeing.”

“I believe we have a moral imperative to find better models to study [psychiatric] conditions,” says Pasca. “Certainly, the more human these models are becoming, the more uncomfortable we feel about these conditions, but I feel that human psychiatric disorders are, to a large extent, uniquely human, so we are going to have to think very carefully together how far we want to go with the models at this point.”

“Minimal concerns”

Lancaster has also considered the moral implications of the study. “I do not have any concerns around whether the human transplants would cause the animal to become more ‘human’ since the size of these transplants are small and their overall organization is still lacking,” she comments.  “Thus, I see this as a model to investigate human neuronal maturation on a single neuron or group of neuron level, but these are not actual brain tissues being implanted and so there are minimal concerns around their potential for higher cognitive functions.”

Could the same technology be used in more complex brains that are more closely related to humans, such as primates? Pasca points out that such an experiment would not only have technological barriers to overcome but would be far more concerning from a moral standpoint. “The transplantations today in rats involves natural barriers to how much integration there can be obtained,” he says, pointing to the wildly different developmental timescales of rats and humans.

“But if the timing of the species that you transplanted in was much closer to humans, you would expect the integration to be much closer,” warns Pasca. “But we don’t consider at this time that [using primates] is necessary. First, we have to leverage the technology we’ve developed, put it to play and see what it can actually teach us about the development of the human brain.”

Reference: Revah O, Gore F, Kelley KW et al. Maturation and circuit integration of transplanted human cortical organoids. Nature. 2022. Doi: 10.1038/s41586-022-05277-w