Cerebral organoids, or mini-brains, developed at the Muotri Lab in UC San Diego’s School of Medicine. Photo: Alysson Renato Muotri

Clusters of brain cells that are barely visible to the naked eye are being grown in labs, and researchers are optimistic that these “mini-brains” could improve the drug discovery process and offer new insight into neurological diseases.

The complexity of the inner workings of the human brain has made studying neurological disorders and diseases one of the biggest challenges for researchers.

Mini-brains, which are essentially tiny balls of human neurons and other cells that mimic some of the brain’s structures and functionality during the earliest stages of development, are grown in the lab over the course of two to three months.

They are produced using induced pluripotent stem cells (iPSCs) or “blank slate cells.” Like the nickname suggests, these adult cells can be genetically reprogrammed to act like embryonic stem cells, and are then stimulated to grow into brain cells. Similar techniques have been done to produce mini-livers and hearts, as well.

More than 100 of the cerebral organoids can grow in the same petri dish or shaker, and they are capable of developing numerous interdependent brain regions seen in real human brains, such as the cerebral cortex.

Mini-brains grown at the Johns Hopkins Bloomberg School of Public Health, for example, developed four types of neurons and two types of support cells, and even showed spontaneous electrophysiological activity.  

“These mini-brains are spontaneously and electrophysiolgically active, so you can monitor their communication in a noninvasive way,” Thomas Hartung, professor of environmental health sciences at the Bloomberg School, told Laboratory Equipment.

An important factor in these specific mini-brains is that the composition doesn’t change from batch to batch, ensuring standardization and accuracy. Since presenting the findings in February 2016, Hartung helped create a spinoff company, Organome, to make the standardized mini-brains commercially available. Organome also received a provisional patent for freezing and thawing the organoids while keeping key features intact.

Hartung admits the freezing and thawing technique still needs to be fine-tuned, but “the business impact is enormous.” The ability to freeze mini-brains would eliminate any potential issues during transport, and would allow the team to freeze and stockpile many mini-brains at a time, instead of continuously having to be in production. This process would allow scientists around the country and world to work with the organoids to further neuroscience research.

“We are very excited to provide a model, which is a problem-solver, that can enable research on human materials, which is typically not possible,” said Hartung.

Why are mini-brains needed?
As the director of the Center for Alternatives to Animal Testing at the Bloomberg School, Hartung’s primary focus is to use mini-brains to replace mouse and other animal models in pharmaceutical testing.

Drug development and testing is a long and expensive trial-and-error process. Currently, the earliest stages of pharmaceutical testing are done in animal models. However, 97 percent of drugs that show promise in mouse or rat models fail in human trials, according to Hartung.   

“Current models aren’t really satisfying. Mouse and rat models of brain diseases have been notorious for not predicting human outcomes,” he said. The human brain is unique, and even primate models don’t accurately portray the effects of neurological diseases in the human brain.

The only way to fully understand what goes wrong in the brain with conditions like Alzheimer’s, Parkinson’s, autism and microphaly is to produce a model grown from live human cells.

According to Hartung, getting a drug on the market just one day earlier can mean an extra $1 million for the drug company, so the monetary impact alone is appealing to drug discovery teams.

“The brain is such an important organ, 25 percent of all clinical trials are for drugs around the central nervous system and side effects of the central nervous system are so key for drugs, but also for pesticides and others,” Hartung noted.

A mini-brain infected with Zika virus. The virus is shown in green, vulnerable neural progenitor cells are shown in red, and neurons are shown in blue. Photo: Xuyu Qian/Johns Hopkins Medicine

One of the most significant advantages of mini-brain models is their wide range of potential applications. They can be used to study virtually any human-specific neurological disorder, and researchers have already made progress with Zika, autism and even orphan diseases.

Hongjun Song, director of the stem cell program at the Institute for Cell Engineering at Johns Hopkins University School of Medicine, and his team found that mini-brain models offered an ideal system to address one of the most urgent health questions of the summer: does Zika cause microcephaly?

Microcephaly is a condition where the brain does not develop properly, resulting in a smaller than normal head and risk of intellectual disability, poor motor function and seizures, among other symptoms. While mouse models failed to provide any valuable insight, Song’s mini-brains helped him discover that the Zika virus targets neural stem cells, which generate the building blocks for the human cortex.

“The consequence of infection is cell death and reduced proliferation, resulting in a thinner cortical layer that mimics microcephaly in the fetus,” explained Song. Song’s team presented these findings at the Society of Neuroscience’s annual meeting in November 2016, and testing of candidate drugs has already begun.

The Muotri Lab, at UC San Diego’s School of Medicine, has also proven how truly versatile mini-brains can be for neuroscientists.
“Some of the evolutionary properties that make us uniquely human and allow us to live the advanced lifestyle we enjoy are also a root cause for a number of disorders. Our brain grants us far greater processing power than any other species, but a complex brain came with a cost: it increases the opportunity for neurological diseases,” write the lab’s researchers.

Alysson Renato Muotri, director of the stem cell program at the Institute for Genomic Medicine, has made headlines recently for unlocking mysteries behind autism, Zika and Cockayne syndrome.

“I want to know ‘what is the human mind’, when consciousness first appears during development and if we can augment the human brain. We try to answer these questions by comparing mini-brains from humans to the ones derived from other primates, such as chimpanzees. At the end, if you understand how the human brain evolved to its current form, you will gain insights on when and why it goes awry, such as in different neurological disorders, providing the opportunity to design better therapeutic interventions,” Muotri explained to Laboratory Equipment.

Thanks to mini-brain models and genome sequencing, Muotri and her team successfully revealed an underlying molecular and cellular pathology that is shared among some individuals with autism. Twenty percent of ASD (autism spectrum disorder) individuals also have macrencephaly, which causes early neuronal overgrowth and abnormally enlarged brains, and precedes the first clinical signs of ASD.
Muotri said the findings show it is possible to more effectively stratify ASD individuals for clinical trials by identifying persons who are likely to be responsive to specific therapies using their mini-brains in a dish.

The team replicated this success to also produce the first human in vitro model of Cockayne syndrome, a rare and debilitating disorder. This allowed researchers to identify areas of cellular dysfunction compared to normal neuronal networks from control models and observe networks that displayed altered electrophysiological activity, including diminished ability to grow synaptic connections to other neurons and synchronize activities.

Animal models for diseases as rare as Cockayne syndrome don’t exist, so without mini-brain models, research would continue to remain at a near-standstill.

Despite the success stories, mini-brain models aren’t ready to fully replace all animal models just yet.

Hartung told Laboratory Equipment that mini-brains stop growing after about 20,000 to 30,000 cells, so they currently only resemble a very young developing human brain. Determining the right protocols and conditions to allow models to resemble later stages of maturation remains an unsolved problem, so diseases that may not present themselves until later in development still remain mysterious.
Additionally, as Muotri pointed out, the technology is still new, so different research groups have different protocols that may achieve varying levels of maturation.

However, this technique, which was thought to be unattainable just a decade ago, is paving a new, more efficient way to conduct neuroscience research.