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Transplanted Human Brain Cells Respond to Visual Stimuli in Rat Brains

An image of a rat brain, where human-derived brain organoids have been transplanted.
This is a histological image of a rat brain with a grafted human brain organoid. Credit: Jgamadze et al.
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A new study by researchers at the University of Pennsylvania has found that human-derived brain organoids can integrate into the visual cortex of rat brains. After three months, the organoids demonstrate electrical responses to visual stimuli. The research is published in Cell Stem Cell.

The utility of organoids in modern medicine

As regulatory agencies such as the United States Food and Drug Administration (FDA) seek to phase out animal testing, organoids – three-dimensional (3D) cell culture systems, sometimes known as “mini-organs” – provide an attractive alternative model for preclinical studies. Now, research suggests they could also support efforts to repair the brain after injury.

What are organoids and how are they produced?

The term “organoid” encompasses 3D cell culture systems that can be derived from stem cells, tumors, tissue explants, stem cells or other progenitor cells. Organoids “self-organize” under specific, controlled conditions, resembling the anatomy, physiology and complexity of organs or other body structures in a dish.

Organoids modeling embryos, tumors, a variety of different tissue types and organs have been generated – including the brain. Brain organoids are created using human pluripotent stem cells (hPSCs), which are generally embedded into an artificial scaffold and cultivated. During the cultivation process, specific growth factors and other molecules are added to the matrix, directing the hPSCs to form specific brain regions or neuronal cell types. Mature brain organoids are regarded as “an excellent model” for studying neuronal development and neurological diseases.  

In 2022, scientists led by Professor Sergiu Pasca at Stanford University transplanted human brain organoids into rat brains. While the concept was not novel, the study was innovative in how the researchers strived to maximize the integration of the organoids. They were transplanted into the somatosensory cortex of young rats at postnatal days three to seven. At this stage of the rat’s life, axonal projections from other brain regions have not yet completed their innovation of the somatosensory cortex. The organoids integrated in 81% of the 72 rats receiving transplants after 2 months, and even responded to stimulation of the rats’ whiskers.

“The work by Pasca’s group was outstanding,” Dr. Han-Chiao Isaac Chen, a physician and assistant professor of Neurosurgery at the University of Pennsylvania says. “They injected organoids into the brains of very young rats that did not have an injury other than the needle being inserted to deliver the organoid. Thus, it is a great model for neurodevelopment.”

Beyond their use as laboratory models, brain organoids also carry potential as an approach for repairing the brain after injury, via implantation of autologous or patient-matched neural tissues. Chen is the lead author of a new study using brain organoids to develop a model of neural repair. They wanted to explore whether human-derived brain organoids can integrate into an injured area of the rat brain, survive and respond to stimuli.  

Transplanting human organoids into the injured rat brain

Chen and colleagues generated forebrain cortical organoids from hPSCs, which were cultivated for 80 days before being transplanted into the visual cortex of 46 adult rats with an injury cavity. The researchers acknowledge this cultivation period is longer than most prior studies: “We chose day 80 organoids primarily because there is more diversity of cell types and more structure resembling the normal brain at this age. Also, older organoids are less likely to overgrow after transplantation, which has been shown in younger organoids in some prior studies,” explains Chen.

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The decision to implant the organoids into the visual cortex is attributed to the many responses that this region of the brain demonstrates to visual stimulation. “These tiers of responses allowed us to interrogate the degree to which organoids had integrated with the host brain,” Chen says. “Also, current protocols for producing neurons and brain organoids from stem cells are biased toward a visual cortex fate. Transplanting a visual cortex organoid into the visual cortex made sense.”

“We focused on adult animals with an injury cavity. The adult brain is thought to be less plastic and with less potential for recovery, but our study showed that organoid grafts can still integrate with the host brain and adopt sophisticated visual cortex functions,” says Chen.

Assessing integration and connectivity

Graft survival was evaluated in the animals at 1, 2 and 3 months post-transplantation, tested in 8, 30 and 8 rats respectively. After 3 months, the overall survival rate of the organoid grafts was 82.1%. Histological and other morphological analyses taken at this timepoint revealed that the organoids had become vascularized, increasing both in size and in quantity.

Transsynaptic tracing methods are adopted in neuroscience to study the organization of neurons’ afferent and efferent connections – i.e., those that project towards a central point (such as the central nervous) and those that project away (e.g., to the periphery). Such methods are enabling scientists to analyze the connectivity between host and grafted neurons faster and at a more intricate level of detail than before.

Chen and colleagues used fluorescent-tagged viruses to assess the synaptic connections between the organoid and the host rats’ own brain cells, revealing extensive efferent and afferent connectivity. “By injecting one of these viral tracers into the eye of the animal, we were able to trace the neuronal connections downstream from the retina,” says Chen. “The tracer got all the way to the organoid.”

“We were not expecting to see this degree of functional integration so early,” says Chen. “There have been other studies looking at transplantation of individual cells that show that even 9 or 10 months after you transplant human neurons into a rodent, they're still not completely mature.”

Transplanted human organoids respond to visual stimuli in the rat brain

Next, using in vivo extracellular recordings, Chen and colleagues assessed the level of electrical activity of neurons within the organoid graft in 10 of the transplanted rats, while exposing them to flashing lights and alternating black and white bars. “Around 20% of the organoid neurons responded to visual stimulation, whereas ~75% or the neurons in the rat’s visual cortex responded to visual stimulation. We did not quantify the number of organoid neurons that responded to orientations of light because this is a continuous – not a binary – variable,” describes Chen.

The researchers’ goal is for the grafted neurons to function as closely as possible to the normal brain cells. “Thus, there were fewer neurons that responded to light than ideal. Understanding how to improve this response rate/integration is one of our primary goals for the future,” adds Chen.

The team regard their proof-of-principle study as a “solid first step”, however they acknowledge it carries some limitations. They assessed the transplantation outcomes after three months. Other studies have demonstrated that the maturation of human neurons derived from stem cells can take approximately ~12 months or longer. “Looking at longer time points is something we are interested in doing in the future,” says Chen.

“A second point is that, while the aspiration cavity we made is a brain injury of sorts, it is not a good model for conditions like traumatic brain injury and stroke. We would like to move our transplantation studies into these types of models in the future,” he adds.

To advance their current work, the team are pursuing the transplantation of organoids into other regions of the cortex. They are also interested in identifying the factors that regulate integration, so that the process can be controlled or perhaps even regulated.

Technology Networks asks Chen how realistic it is that neural tissues – such as organoids – could be used to repair brain injuries in the future. “Many parts of the brain are highly structured with repetitive units. The cerebral cortex is like this. It has functional units called ‘columns’ that are thought to be the fundamental unit of processing in the cortex,” Chen says, concluding that “repairing the cortex in the future could take the form of inserting additional ‘engineered cortical columns’ into the injured brain to increase its computational capacity.”

Dr. Han-Chiao Isaac Chen was speaking to Molly Campbell, Senior Science Writer for Technology Networks.

Reference: Jgamadze D, Lim J, Zhang Z, et al. Structural and functional integration of human forebrain organoids with the injured adult rat visual system. Cell Stem Cell. 2023. doi: 10.1016/j.stem.2023.01.004