Stem cell stories that caught our eye: the tale of a tail that grows back and Zika’s devious Trojan Horse

The tale of a tail that grows back (Kevin McCormack)

Ask people what they know about geckos and the odds are they’ll tell you geckos have English accents and sell car insurance. Which tells you a lot more about the power of advertising than it does about the level of knowledge about lizards. Which is a shame, because the gecko has some amazing qualities, not the least of which is its ability to re-grow its tail. Now some researchers have discovered how it regenerates its tail, and what they’ve learned could one day help people with spinal cord injuries.

Geckos often detach a bit of their tail when being pursued by a predator, then grow a new one over the course of 30 days. Researchers at the University of Guelph in Canada found that the lizards use a combination of stem cells and proteins to do that.

They found that geckos have stem cells in their tail called radial glias. Normally these cells are dormant but that changes when the lizard loses its tail. As Matthew Vickaryous, lead author of the study, said in a news release:

“But when the tail comes off everything temporarily changes. The cells make different proteins and begin proliferating more in response to the injury. Ultimately, they make a brand new spinal cord. Once the injury is healed and the spinal cord is restored, the cells return to a resting state.”

Vickaryous hopes that understanding how the gecko can repair what is essentially an injury to its spinal cord, we’ll be better able to develop ways to help people with the same kind of injury.

The study is published in the Journal of Comparative Neurology.

Zika virus uses Trojan Horse strategy to infect developing brain
In April 2015, the World Health Organization declared that infection by Zika virus and its connection to severe birth defects was an international public health emergency. The main concern has been the virus’ link to microcephaly, a condition in which abnormal brain development causes a smaller than normal head size at birth. Microcephaly leads to number of problems in these infants including developmental delays, seizures, hearing loss and difficulty swallowing.

A false color micrograph shows microglia cells (green) infected by the Zika virus (blue). Image Muotri lab/UCSD

Since that time, researchers have been racing to better understand how Zika infection affects brain development with the hope of finding treatment strategies. Now, a CIRM-funded study in Human Molecular Genetics reports important new insights about how Zika virus may be transmitted from infected pregnant women to their unborn babies.

The UCSD researchers behind the study chose to focus on microglia cells. In a press release, team leader Alysson Muotri explained their rationale for targeting these cells:

“During embryogenesis — the early stages of prenatal development — cells called microglia form in the yolk sac and then disperse throughout the central nervous system (CNS) of the developing child. Considering the timing of [Zika] transmission, we hypothesized that microglia might be serving as a Trojan horse to transport the virus during invasion of the CNS.”

In the developing brain, microglia continually travel throughout the brain and clear away dead or infected cells. Smuggling itself aboard microglia would give Zika a devious way to slip through the body’s defenses and infect other brain cells. And that’s exactly what Dr. Muotri’s team found.

Using human induced pluripotent stem cells (iPSCs), they generated brain stem cells – the kind found in the developing brain – and in lab dish infected them with Zika virus. When iPSC-derived microglia were added to the infected neural stem cells, the microglia gobbled them up and destroyed them, just as they would do in the brain. But when those microglia were placed next to uninfected brain stem cells, the Zika virus was easily transmitted to those cells. Muotri summed up the results this way:

“Our findings show that the Zika virus can infect these early microglia, sneaking into the brain where they transmit the virus to other brain cells, resulting in the devastating neurological damage we see in some newborns.”

The team went on to show that an FDA-approved drug to treat hepatitis – a liver disease often caused by viral infection – was effective at decreasing the infection of brain stem cells by Zika-carrying microglia. Since these studies were done in petri dishes, more research will be required to confirm that the microglia are a true drug target for stopping the devastating impact of Zika on newborns.

Taming the Zika virus to kill cancer stem cells that drive lethal brain tumor

An out of control flame can be very dangerous, even life-threatening. But when harnessed, that same flame sustains life in the form of warm air, a source of light, and a means to cook.

A similar duality holds true for viruses. Once it infects the body, a virus can replicate like wildfire and cause serious illness and sometimes death. But in the lab, researchers can manipulate viruses to provide an efficient, harmless method to deliver genetic material into cells, as well as to prime the immune system to protect against future infections.

In a Journal of Experimental Medicine study published this week, researchers from the University of Washington, St. Louis and UC San Diego also show evidence that a virus, in this case the Zika virus, could even be a possible therapy for a hard-to-treat brain cancer called glioblastoma.

Brain cancer stem cells (left) are killed by Zika virus infection (image at right shows cells after Zika treatment). Image: Zhe Zhu, Washington U., St. Louis.

Recent outbreaks of the Zika virus have caused microcephaly during fetal development. Babies born with microcephaly have a much smaller than average head size due to a lack of proper brain development. Children born with this condition suffer a wide range of devastating symptoms like seizures, difficulty learning, and movement problems just to name a few. In the race to understand the outbreak, scientists have learned that the Zika virus induces microcephaly by infecting and killing brain stem cells, called neural progenitors, that are critical for the growth of the developing fetal brain.

Now, glioblastoma tumors contain a small population of cells called glioblastoma stem cells (GSCs) that, like neural progenitors, can lay dormant but also make unlimited copies of themselves.  It’s these properties of glioblastoma stem cells that are thought to allow the glioblastoma tumor to evade treatment and grow back. The research team in this study wondered if the Zika virus, which causes so much damage to neural progenitors in developing babies, could be used for good by infecting and killing cancer stem cells in glioblastoma tumors in adult patients.

To test this idea, the scientists infected glioblastoma brain tumor samples with Zika and showed that the virus spreads through the cells but primarily kills off the glioblastoma stem cells, leaving other cells in the tumor unscathed. Since radiation and chemotherapy are effective at killing most of the tumor but not the cancer stem cells, a combination of Zika and standard cancer therapies could provide a knockout punch to this aggressive brain cancer.

Even though Zika virus is much more destructive to the developing fetal brain than to adult brains, it’s hard to imagine the US Food and Drug Administration ever approving the injection of a dangerous virus into the site of a glioblastoma tumor. So, the scientists genetically modified the Zika virus to make it more sensitive to the immune system’s first line of defense called the innate immunity. With just the right balance of genetic alterations, it might be possible to retain the Zika virus’ ability to kill off cancer stem cells without causing a serious infection.

The results were encouraging though not a closed and shut case: when glioblastoma cancer stem cells were infected with these modified Zika virus strains, the virus’ cancer-killing abilities were weaker than the original Zika strains but still intact. Based on these results, co-senior author and WashU professor, Dr. Michael S. Diamond, plans to make more tweaks to the virus to harness it’s potential to treat the cancer without infecting the entire brain in the process.

“We’re going to introduce additional mutations to sensitize the virus even more to the innate immune response and prevent the infection from spreading,” said Diamond in a press release. “Once we add a few more changes, I think it’s going to be impossible for the virus to overcome them and cause disease.”

 

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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Timing Matters: Slowly Dividing Stem Cells Lead to Small Brains

One hundred billion nerve cells working together empowering us to see, walk, think, speak, remember: the human brain is a stunning machine. Even more stunning is its formation in the growing fetus. It starts with a set of neural, or brain, stem cells in the early embryo. Then with each cell division, more and more cells emerge and specialize to perform various brain functions. All the while, some of those “daughter” cells remain uncommitted, staying in their neural stem cell form in order to keep a steady cell supply to build the brain. With all these 100,000,000,000 cells needing to be assembled in a precise way,  it’s amazing that our brains work at all.

Microcephaly

MRIs of a healthy individual (left) and a patient with microcephaly (right). Credit: PLoS Biol 2(5): e134

Suffer the children
Well, sometimes they don’t. For about 25,000 babies born in the U.S. each year, the brain doesn’t grow the way it should, leading to microcephaly, a disorder characterized by an abnormally small head (micro=small; cephaly=head). These babies have a range of symptoms including speech delays, seizures, mental retardation and balance difficulties.

Preventing microcephaly first requires understanding why this devastating condition leads to a smaller brain size. On Thursday, a Duke research team reported in Neuron that they have the answer: stem cells dividing too slowly.

In 2010, the team, led by Debra Silver, an assistant professor of molecular genetics and microbiology at the Duke University School of Medicine, found that mice lacking one of two copies of the Magoh gene showed a reduced brain size much like what’s seen in human microcephaly. Their study showed that the genetic defect upset the normal ratio of neural stem cells to neurons (nerve cells) in the brain. But how?

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Slowly dividing neural stem cells generate aberrant stem cells and neurons. Credit: Louis-Jan Pilaz, Duke University

That’s where this new data comes into play. They found that many neural stem cells in mice lacking one copy of the Magoh gene divided at a slower rate, taking up to three times longer than cells with both copies of Magoh. Using specialized microscopes, the team observed the cells in real time and noticed that the slowly dividing cells were more likely to specialize into neurons rather remain in a stem cell state. On top of that, these neurons died off more readily. Silver described the results of this double-whammy of defects in a Duke University press release:

“It’s really a combination that helps explain the microcephaly. On one hand, you’re really not making enough new stem cells, and if you don’t have enough stem cells you can’t make enough neurons in the brain. On the other hand, some neurons do get made, but a lot of them die.”

Proving their point
To back up this claim, the team treated healthy mice with drugs that lengthen the time it takes for a cell to divide. Sure enough, an unusually high number of neural stem cells from those mice specialized into neurons and then succumbed to an early death.

From Silver’s perspective, this discovery doesn’t just provide a foothold in understanding (and maybe in even one day treating) microcephaly, it could be a fundamental insight for human developmental disorders in general:

This study shows that the time it takes for a stem cell to divide matters during brain development, But beyond microcephaly, I think it’s going to be relevant for thinking about how stem cell dysfunction can change the repertoire of other cells in the body.