Brain Models Get an Upgrade: 3D Mini-Brains

Every year, companies like Apple, Microsoft and Google work tirelessly to upgrade their computer, software and smartphone technologies to satisfy growing demands for more functionality. Much like these companies, biomedical scientists work tirelessly to improve the research techniques and models they use to understand and treat human disease.

Today, I’ll be talking about a cool stem cell technology that is an upgrade of current models of neurological diseases. It involves growing stem cells in a 3D environment and turning them into miniature organs called organoids that have similar structures and functions compared to real organs. Scientists have developed techniques to create organoids for many different parts of the body including the brain, gut, lungs and kidneys. These tiny 3D models are useful for understanding how organs are formed and how viruses or genetic mutations can affect their development and ability to function.

Brain Models Get an Upgrade

Organoids are especially useful for modeling complex neurological diseases where current animal and 2D cell-based models lack the ability to fully represent the cause, nature and symptoms of a disease. The first cerebral, or brain, organoids were generated in 2013 by Dr. Madeline Lancaster in Austria. These “mini-brains” contained nerve cells and structures found in the cortex, the outermost layer of the human brain.

Since their inception, mini-brains have been studied to understand brain development, test new drugs and dissect diseases like microcephaly – a disease that causes abnormal brain development and is characterized by very small skulls. Mini-brains are still a new technology, and the question of whether these organoids are representative of real human brains in their anatomy and behavior has remained unanswered until now.

Published today in Cell Reports, scientists from the Salk Institute reported that mini-brains are more like human brains compared to 2D cell-based models where brain cells are grown in a single layer on a petri dish. To generate mini-brains, they collaborated with a European team that included the Lancaster lab. They grew human embryonic stem cells in a 3D environment with a cocktail of chemicals that prompted them to develop into brain tissue over a two-month period.

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

After generating the mini-brains, the next step was to prove that these organoids were an upgrade for modeling brain development. The teams found that the cells and structures formed in the mini-brains were more like human brain tissue at the same stage of early brain development than the 2D models.

Dr. Juergen Knoblich, co-senior author of the new paper and head of the European lab explained in a Salk News Release, “Our work demonstrates the remarkable degree to which human brain development can be recapitulated in a dish in cerebral organoids.”

Are Mini-Brains the Real Thing?

The next question the teams asked was whether mini-brains had similar functions and behaviors to real brains. To answer that question, the scientists turned to epigenetics. This is a fancy word for the study of chemical modifications that influence gene expression without altering the DNA sequence in your genome. The epigenome can be thought of as a set of chemical tags that help coordinate which genes are turned on and which are turned off in a cell. Epigenetics plays important roles in human development and in causing certain diseases.

The Salk team studied the epigenomes of cells in the mini-brains to see whether their patterns were similar to cells found in human brain tissue. Interestingly, they found that the epigenetic patterns in the 3D mini-brains were not like those of real brain tissue at the same developmental stage. Instead they shared a commonality with the 2D brain models and had random epigenetic patterns. While the reason for these results is still unknown, the authors explained that it is common for cells and tissues grown in a lab dish to have these differences.

In a Salk news release, senior author and Salk professor Dr. Joseph Ecker said that even though the current mini-brain models aren’t perfect yet, scientists can still gather valuable information from them in the meantime.

“Our findings show that cerebral organoids as a 3D model of brain function are getting closer to a real brain than 2D models, so perhaps by using the epigenetic pattern as a gauge we can get even closer.”

And while the world eagerly waits for the next release of the iPhone 7, neuroscientists will be eagerly waiting for a new and improved version of mini-brains. Hopefully the next upgrade will produce organoids that behave more like the real thing and can model complex neurological diseases, such as Alzheimer’s, where so many questions remain unanswered.

UCSD scientists find new clue into how Zika virus impairs brain development

In April of this year, the Centers for Disease Control and Prevention (CDC) announced their conclusion that Zika virus causes microcephaly, a birth defect that results in abnormal brain development and a smaller sized head in infants. Rather than a single study being responsible for their conclusion, the CDC argued that “mounting evidence” from multiple recent reports has made the link between Zika infection in pregnant women and microcephaly undeniable.

Now that the general consensus is that Zika virus impairs brain development, scientists are making fast progress to develop appropriate models of brain development to understand exactly how the virus causes microcephaly. We recently blogged about one study from UC San Francisco, which found a molecular link between Zika infection and the function of brain stem cells. They used a brain organoid model, derived from human stem cells, to identify a protein receptor called AXL that is expressed on the surface of brain stem cells and is a major entry point for Zika virus infection.

The power of mini-brains

Cross section of a brain organoid. (MIT Tech Review)

Cross section of a brain organoid. (MIT Tech Review)

So called “mini-brains”, or 3D brain organoids, have proven to be a very useful model for brain development and Zika virus infection. With rapid advances in stem cell technologies, mini-brains now develop the appropriate cell types and brain structures representative of the first trimester of fetal brain development. They also can be derived from both embryonic stem cells and induced pluripotent stem cells, making them a versatile technology that can model patient specific diseases.

Speaking of mini-brains, a study was published just last week in the journal Cell Stem Cell from UC San Diego that used mini-brains to identify an immune system molecule that gets hijacked by the Zika virus. They found that Toll-like-Receptor 3 (TLR3) negatively impacts the ability of brain stem cells to differentiate or specialize into the mature cells of the brain.

When the organoids were exposed to a strain of the Zika virus, MR766, their size five days later was smaller than organoids that weren’t exposed to the virus. The growth rate for normal organoids in the time period was 22.6% while the rate for Zika-treated organoids was only 16%. Dissection of the Zika-treated organoids revealed that the virus was successful in infecting brain stem cells specifically and somehow impaired their ability to differentiate. They also noticed that a specific immune molecule called TLR3 was abnormally activated in the organoids after Zika infection.

TLR3: too much of a good thing

In an attempt to put the puzzle pieces together, the authors focused on TLR3 and its potential role in causing brain development defects caused by Zika virus. TLR3 is a sentinel of the innate immune system, the body’s first line defense against infection. It’s a receptor on the surface of cells that can recognize foreign viruses and mount an immune response by activating infection fighting genes.

Brain organoids were used to model Zika virus infection. (Cell Stem Cell)

Brain organoids were used to model Zika virus infection. (Cell Stem Cell)

TLR3 sounds like a good guy when it comes to defending the immune system, but there are cases where too much TLR3 is not a good thing. Activation of TLR3 in Zika-infected brain organoids turned on a group of 41 genes that blocked the differentiation of brain stem cells, causing brain organoid shrinkage, and also caused the stem cells to commit apoptosis, a cellular form of programmed suicide.

Logically, the authors tested whether blocking the activity of TLR3 in Zika-infected organoids alleviated these negative effects. A TLR3 inhibitor was effective at preventing brain stem cell apoptosis and also organoid shrinkage in Zika-treated organoids. However, the treatment wasn’t perfect, the Zika-infected organoids did not grow to the same size as untreated organoids after TLR3 inhibition and still experienced more cell death.

Senior author on the study Dr. Tariq Rana explained:

“We all have an innate immune system that evolved specifically to fight off viruses, but here the virus turns that very same defense mechanism against us. By activating TLR3, the Zika virus blocks genes that tell stem cells to develop into the various parts of the brain. The good news is that we have TLR3 inhibitors that can stop this from happening.”

The size of brain organoids is reduced with Zika infection but partly rescued with a TLR3 inhibitor. Normal (left), Zika infected (middle), Zika infected with TLR3 inhibitor treatment. (Cell Stem Cell)

The size of brain organoids is reduced with Zika infection but partly rescued with a TLR3 inhibitor. Normal (left), Zika infected (middle), Zika infected with TLR3 inhibitor treatment. (Cell Stem Cell)

Next Steps

In a UCSD press release, the authors admit that this work is still in its early stages. The experiments they conducted used both mouse and human cells and further work is needed to determine whether TLR3 is an appropriate target for blocking Zika infection in humans.

They also note that this study tests only one strain of the Zika virus, one that originated in Uganda, and that other strains prevalent in countries like Latin America and Asia should be tested as well. Other strains could have different mechanisms of infection and different effects on the function of brain stem cells.

Rana acknowledged this and commented:

Dr. Tariq Rana, UCSD

Dr. Tariq Rana, UCSD

“We used this 3D model of early human brain development to help find one mechanism by which Zika virus causes microcephaly in developing fetuses, but we anticipate that other researchers will now also use this same scalable, reproducible system to study other aspects of the infection and test potential therapeutics.”

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UCSF Scientists find molecular link between brain stem cells and Zika Infection

The Zika virus scare came to a head in 2015, prompting the World Health Organization to declare the outbreak a global health emergency earlier this year. From a research standpoint, much of the effort has centered on understanding whether the Zika infection is actually a cause of birth defects like microcephaly and how the virus infects mothers and their unborn children.

The Zika Virus is spread by a specific type of mosquito, the Aedes aegypti.

The Zika Virus is spread to humans by mosquitos.

What’s known so far is that the Zika virus can pass from the mother to the fetus through the placenta and it can infect the developing brain of the fetus. But how exactly the virus infects brain cells is less clear.

Brain stem cells are vulnerable to Zika

Scientists from UC San Francisco (UCSF) are tackling this question and have unraveled one more piece to the Zika infection puzzle. UCSF professor Dr. Arnold Kriegstein and his team reported yesterday in the journal Cell Stem Cell that they’ve identified a protein receptor on the surface of brain stem cells that could be the culprit for Zika virus infection.

Based on previous studies that showed that the Zika virus specifically infects brain stem cells, Kriegstein and his colleagues hypothesized that these cells expressed specific proteins that made them vulnerable to Zika infection. They looked to see which genes were turned on and off in brain stem cells derived from donated fetal tissue as well as other cell types in the developing brain to identify proteins that would mediate Zika virus entry.

AXL is to blame

They found a protein receptor called AXL that was heavily expressed in a type of brain stem cell called the radial glial cell, which gives rise to the outer layer of the brain called the cerebral cortex. AXL piqued their interest because it was identified in other studies as an entry point for Zika and other similar viruses like dengue in human skin cells. Furthermore, the team confirmed that radial glial cells produce a lot of AXL protein during development and it appears during a specific window of time – the second trimester of pregnancy.

A link between radial glial cells and Zika infection made sense to first author Tomasz Nowakowski who explained in a UCSF news release,

“In the rare cases of congenital microcephaly, these [radial glial cells] are the cells that die or differentiate prematurely, which is one of the reasons we became interested in the possible link.”

The team also found that AXL was expressed in mature brain cells including astrocytes and microglia and in retinal progenitor cells in the eye. They pointed out that the presence of AXL in the developing eye could help explain why many cases of Zika infection are associated with eye defects.

Modeling Zika infection using mini-brains

The bulk of the study used stem cells isolated from donated human fetal tissue, but the team also developed a different stem cell model to confirm their results. They generated brain organoids, also coined as “mini-brains”, in a dish from human induced pluripotent stem cells. These mini-brains contain structures and cell types that closely resemble parts of the developing brain. The team studied radial glial like cells in the mini-brains and found that they also expressed AXL on their surface.

An image of tissue that’s grown in a dish shows radial glia stem cells that are red, neurons in blue and the AXL receptor in green. Photo by Elizabeth DiLullo

Mini-brains grown in a dish have radial glia stem cells (red), neurons (blue) and the AXL receptor (green). Photo by Elizabeth DiLullo, UCSF

Kriegstein and his team believe they now have a working stem cell model for how viruses like Zika can infect the brain. Using their brain organoid model, they plan to collaborate with other UCSF researchers to learn more about how Zika infection occurs and whether it really causes birth defects.

“If we can understand how Zika may be causing birth defects,” Kriegstein said, “we can start looking for compounds to protect pregnant women who become infected.”

What’s next?

While the evidence points towards AXL as one of the major entry points for Zika infection in the developing brain, the UCSF team and other scientists still need to confirm that this receptor is to blame.

Kriegstein explained:

Arnold Kriegstein, UCSF

Arnold Kriegstein, UCSF

“While by no means a full explanation, we believe that the expression of AXL by these cell types is an important clue for how the Zika virus is able to produce such devastating cases of microcephaly, and it fits very nicely with the evidence that’s available. AXL isn’t the only receptor that’s been linked with Zika infection, so next we need to move from ‘guilt by association’ and demonstrate that blocking this specific receptor can prevent infection.”

If AXL turns out to be the culprit, scientists will have to be careful about testing drugs that block its function given that AXL is important for the proliferation of brain stem cells during development. There might be a way however that such treatments could be given to at risk women before they get pregnant.

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