Stem Cell Roundup: The brain & obesity; iPSCs & sex chromosomes; modeling mental illness

Stem Cell Image of the Week:
Obesity-in-a-dish reveals mutations and abnormal function in nerve cells

cedars-sinai dayglo

Image shows two types of hypothalamic neurons (in magenta and cyan) that were derived from human induced pluripotent stem cells.
Credit: Cedars-Sinai Board of Governors Regenerative Medicine Institute

Our stem cell image of the week looks like the work of a pre-historic cave dweller who got their hands on some DayGlo paint. But, in fact, it’s a fluorescence microscopy image of stem cell-derived brain cells from the lab of Dhruv Sareen, PhD, at Cedars-Sinai Medical Center. Sareen’s team is investigating the role of the brain in obesity. Since the brain is a not readily accessible organ, the team reprogrammed skin and blood cell samples from severely obese and normal weight individuals into induced pluripotent stem cells (iPSCs). These iPSCs were then matured into nerve cells found in the hypothalamus, an area of the brain that regulates hunger and other functions.

A comparative analysis showed that the nerve cells derived from the obese individuals had several genetic mutations and had an abnormal response to hormones that play a role in telling our brains that we are hungry or full. The Cedars-Sinai team is excited to use this obesity-in-a-dish system to further explore the underlying cellular changes that lead to excessive weight gain. Ultimately, these studies may reveal ways to combat the ever-growing obesity epidemic, as Dr. Sareen states in a press release:

“We are paving the way for personalized medicine, in which drugs could be customized for obese patients with different genetic backgrounds and disease statuses.”

The study was published in Cell Stem Cell

Differences found in stem cells derived from male vs female.

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Microscope picture of a colony of iPS cells. Credit: Vincent Pasque

Scientists at UCLA and KU Leuven University in Belgium carried out a study to better understand the molecular mechanisms that control the process of reprogramming adult cells back into the embryonic stem cell-like state of induced pluripotent stem cells (iPSCs). Previous studies have shown that female vs male embryonic stem cells have different patterns of gene regulation. So, in the current study, male and female cells were analyzed side-by-side during the reprogramming process.  First author Victor Pasquale explained in a press release that the underlying differences stemmed from the sex chromosomes:

In a normal situation, one of the two X chromosomes in female cells is inactive. But when these cells are reprogrammed into iPS cells, the inactive X becomes active. So, the female iPS cells now have two active X chromosomes, while males have only one. Our results show that studying male and female cells separately is key to a better understanding of how iPS cells are made. And we really need to understand the process if we want to create better disease models and to help the millions of patients waiting for more effective treatments.”

The CIRM-funded study was published in Stem Cell Reports.

Using mini-brains and CRISPR to study genetic linkage of schizophrenia, depression and bipolar disorder.

If you haven’t already picked up on a common thread in this week’s stories, this last entry should make it apparent: iPSC cells are the go-to method to gain insight in the underlying mechanisms of a wide range of biology topics. In this case, researchers at Brigham and Women’s Hospital at Harvard Medical School were interested in understanding how mutations in a gene called DISC1 were linked to several mental illnesses including schizophrenia, bipolar disorder and severe depression. While much has been gleaned from animal models, there’s limited knowledge of how DISC1 affects the development of the human brain.

The team used human iPSCs to grow cerebral organoids, also called mini-brains, which are three-dimensional balls of cells that mimic particular parts of the brain’s anatomy. Using CRISPR-Cas9 gene-editing technology – another very popular research tool – the team introduced DISC1 mutations found in families suffering from these mental disorders.

Compared to cells with normal copies of the DISC1 gene, the mutant organoids showed abnormal structure and excessive cell signaling. When an inhibitor of that cell signaling was added to the growing mutant organoids, the irregular structures did not develop.

These studies using human cells provide an important system for gaining a better understanding of, and potentially treating, mental illnesses that victimize generations of families.

The study was published in Translation Psychiatry and picked up by Eureka Alert.

UC Davis researchers make stem cell-derived mini-brains that contain blood vessels

Growing neurons on a flat petri dish is a great way to study the inner workings of nerve signals in the brain. But I think it’s safe to argue that a two-dimensional lawn of cells doesn’t capture all the complexity of our intricate, cauliflower-shaped brains. Then again, cracking open the skulls of living patients is also not a viable path for fully understanding the molecular basis of brain disorders.

two-spheroids-in-a-dish

Brain organoids (two white balls) growing in petri dish.
Image: Pasca Lab, Stanford University.

The recent emergence of stem cell-derived mini-brains, or brain organoids, as a research tool is bridging this impasse. With induced pluripotent stem cells (iPSCs) derived from a readily-accessible skin sample from patients, it’s possible to generate three-dimensional balls of cells that mimic particular parts of the brain’s anatomy. These mini-brains have the expected type of neurons, as well as other cells that support neuron function. We’ve written many blogs, most recently in January, on the applications of this cutting-edge tool.

With any new technology, there is always room for improvement. One thing that most mini-brains lack is their own system of blood vessels, or vasculature. That’s where Dr. Ben Waldau, a vascular neurosurgeon at UC Davis Medical Center, and his lab come into the picture. Last week, their published work in NeuroReport showed that incorporating blood vessels into a brain organoid is possible.

UCDavisorganoid

A stained cross-section of a brain organoid showing that blood vessels (in red) have penetrated both the outer, more organized layers and the inner core. Image: UC Davis Institute for Regenerative Cures

Using iPSCs from one patient, the Waldau team separately generated brain organoids and blood vessels cells, also called endothelial cells. After growing each for about a month, the organoids were embedded in a gelatin containing the endothelial cells. In an excellent Wired article, writer Megan Molteni explains what happened next:

“After incubating for three weeks, they took a single organoid and transplanted it into a tiny cavity carefully carved into a mouse’s brain. Two weeks later the organoid was alive, well—and, critically, had grown capillaries that penetrated all the way to its inner layers.”

Every tissue relies on nutrients and oxygen from the blood. As Molteni suggests, being able to incorporate blood vessels and brain organoids from the same patient’s cells may make it possible to grow and study even more complex brain structures without the need of a mouse using fluidic pumps.

As Waldau explains in the Wired article, this vascularized brain organoid system also adds promise to the ultimate goal of repairing damaged brain tissue:

waldau

Ben Waldau

“The whole idea with these organoids is to one day be able to develop a brain structure the patient has lost made with the patient’s own cells. We see the injuries still there on the CT scans, but there’s nothing we can do. So many of them are left behind with permanent neural deficits—paralysis, numbness, weakness—even after surgery and physical therapy.”

 

 

Stem Cell Stories that Caught our Eye: Mini-Brains in the Spotlight

Here are the stem cell stories that caught our eye this week.

Two research photos really caught my eye this week and they happened to be of the same thing – mini-brains. Also referred to as brain organoids, mini-brains are tiny balls of nervous tissue grown from stem cells in the lab. They allow scientists to model early brain development and study how disease affects brain cells. Another awesome thing about mini-brains is how cool they look under a microscope.

Mini Brains Part 1

Mini-brain grown in a culture dish. (Photo by Collin Edington and Iris Lee, MIT)

I discovered the first photo in a blog by Dr. Francis Collins, the Director of the National Institutes of Health. He was featuring one of the winning images from the 2017 Koch Institute Image Awards at MIT. The mini-brain photo was taken by researchers Collin Edington and Iris Lee and took over 12 hours to make. Talk about dedication!

Collins revealed that growing mini-brains from stem cells is just the tip of the iceberg for this MIT team. The researchers have plans to grow other types of mini-organs and eventually combine them to make a “human on a chip”. This multi-organ technology will be extremely valuable for studying complex diseases like Alzheimer’s and Parkinson’s, which affect multiple systems in the body.

Mini Brains Part 2

Mini-brain. (Photo by Robert Krencik and Jessy Van Asperen)

The second photo of mini-brains is from a study published this week in Stem Cell Reports by researchers at the Houston Methodist Research Institute. The team has developed a more efficient and effective method for growing mini-brains from stem cells. Typically, the process takes weeks to grow the organoids and months to mature those organoids to the point where they develop the specific cell types and structures found in the human brain.

The Houston team found that maturing different types of brain cells from pluripotent stem cells separately and then combining these mature cells together produced mini-brains that more accurately represented the complexity of the human brain. The trick was to add the brain’s support cells, called astrocytes, to the mini-brains. The astrocytes effectively “accelerated the connections of the surrounding neurons.”

The studies first author, Robert Krencik, explained in a news release,

“We always felt like what we were doing in the lab was not precisely modeling how the cells act within the human brain. So, for the first time, when we put these cells together systematically, they dramatically changed their morphological complexity, size and shape. They look like cells as you would see them within the human brain, so now we can study cells in the lab in a more natural environment.”

Their method also cuts down the time it takes to make mini-brains which will hugely benefit neuroscience researchers who have passed on using mini-brains in their studies because of the cost and time it takes to grow them. Krencik explained,

“Normally, growing these 3-D mini brains takes months and years to develop. We have new techniques to pre-mature the cells separately and then combine them, and we found that within a few weeks they’re able to form mature interactions with each other. So, the length of time to get to that endpoint for studies is dramatically reduced with our system.”

The team plans to use this method to make patient-specific mini-brains from induced pluripotent stem cells to gain new insights into how disease affects the brain. They also hope to translate their mini-brain system into clinical trials to help patients regenerate brain damage or repair brain function.

Stem cell stories that caught our eye: brains, brains and more brains!

This week we bring you three separate stories about the brain. Two are exciting new advances that use stem cells to understand the brain and the third is plain creepy.

Bioengineering better brains. Lab grown mini-brains got an upgrade thanks to a study published this week in Nature Biotechnology. Mini-brains are tiny 3D organs that harbor similar cell types and structures found in the human brain. They are made from pluripotent stem cells cultured in laboratory bioreactors that allow these cells to mature into brain tissue in the span of a month.

The brain organoid technology was first published back in 2013 by Austrian scientists Jürgen Knoblich and Madeline Lancaster. They used mini-brains to study human brain development and a model a birth defect called microcephaly, which causes abnormally small heads in babies. Mini-brains filled a void for scientists desperate for better, more relevant models of human brain development. But the technology had issues with consistency and produced organoids that varied in size, structure and cell type.

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

Fast forward four years and the same team of scientists has improved upon their original method by adding a bioengineering technique that will generate more consistent mini-brains. Instead of relying on the stem cells to organize themselves into the proper structures in the brain, the team developed a biological scaffold made of microfilaments that guides the growth and development of stem cells into organoids. They called these “engineered cerebral organoids” or enCORs for short.

In a news feature on IMBA, Jürgen Knoblich explained that enCORs are more reproducible and representative of the brain’s architecture, thus making them more effective models for neurological and neurodevelopmental disorders.

“An important hallmark of the bioengineered organoids is their increased surface to volume ratio. Because of their improved tissue architecture, enCORs can allow for the study of a broader array of neurological diseases where neuronal positioning is thought to be affected, including lissencephaly (smooth brain), epilepsy, and even autism and schizophrenia.”

Salk team finds genetic links between brain’s immune cells and neurological disorders. (Todd Dubnicoff)

Dysfunction of brain cells called microglia have been implicated in a wide range of neurologic disorders like Alzheimer’s, Parkinson’s, Huntington’s, autism and schizophrenia. But a detailed examination of these cells has proved difficult because they don’t grow well in lab dishes. And attempts to grow microglia from stem cells is hampered by the fact that the cell type hasn’t been characterized enough for researchers to know how to distinguish it from related cell types found in the blood.

By performing an extensive analysis of microglia gene activity, Salk Institute scientists have now pinpointed genetic links between these cells and neurological disease. These discoveries also demonstrate the importance of the microglia’s environment within the brain to maintain its identity. The study results were reported in Science.

Microglia are important immune cells in the brain. They are related to macrophages which are white blood cells that roam through the body via the circulatory system and gobble up damaged or dying cells as well as foreign invaders. Microglia also perform those duties in the brain and use their eating function to trim away faulty or damage nerve connections.

To study a direct source of microglia, the team worked with neurosurgeons to obtain small samples of brain tissue from patients undergoing surgery for epilepsy, a tumor or stroke. Microglia were isolated from healthy regions of brain tissue that were incidentally removed along with damaged or diseased brain tissue.

Salk and UC San Diego scientists conducted a vast survey of microglia (pictured here), revealing links to neurodegenerative diseases and psychiatric illnesses. (Image: Nicole Coufal)

A portion of the isolated microglia were immediately processed to take a snap shot of gene activity. The researchers found that hundreds of genes in the microglia had much higher activities compared to those same genes in macrophages. But when the microglia were transferred to petri dishes, gene activity in general dropped. In fact, within six hours of tissue collection, the activity of over 2000 genes in the cells had dropped significantly. This result suggests the microglial rely on signals in the brain to stimulate their gene activity and may explain why they don’t grow well once removed from that environment into lab dishes.

Of the hundreds of genes whose activity were boosted in microglia, the researchers tracked down several that were linked to several neurological disorders. Dr. Nicole Coufal summarized these results and their implications in a Salk press release:

“A really high proportion of genes linked to multiple sclerosis, Parkinson’s and schizophrenia are much more highly expressed in microglia than the rest of the brain. That suggests there’s some kind of link between microglia and the diseases.”

Future studies are needed to explain the exact nature of this link. But with these molecular descriptions of microglia gene activity now in hand, the researchers are in a better position to study microglia’s role in disease.

A stem cell trial to bring back the dead, brain-dead that is. A somewhat creepy stem cell story resurfaced in the news this week. A company called Bioquark in Philadelphia is attempting to bring brain-dead patients back to life by injecting adult stem cells into their spinal cords in combination with other treatments that include protein blend injections, electrical nerve stimulation and laser therapy. The hope is that this combination stem cell therapy will generate new neurons that can reestablish lost connections in the brain and bring it back to life.

Abstract image of a neuron. (Dom Smith/STAT)

You might wonder why the company is trying multiple different treatments simultaneously. In a conversation with STAT news, Bioquark CEO Ira Pastor explained,

“It’s our contention that there’s no single magic bullet for this, so to start with a single magic bullet makes no sense. Hence why we have to take a different approach.”

Bioquark is planning to relaunch a clinical trial testing its combination therapy in Latin America sometime this year. The company previously attempted to launch its first trial in India back in April of 2016, but it never got off the ground because it failed to get clearance from India’s Drug Controller General.

STATnews staff writer Kate Sheridan called the trial “controversial” and raised questions about how it would impact patients and their families.

“How do researchers complete trial paperwork when the person participating is, legally, dead? If the person did regain brain activity, what kind of functional abilities would he or she have? Are families getting their hopes up for an incredibly long-shot cure?”

Scientists also have questions mainly about whether this treatment will actually work or is just a shot in the dark. Adding to the uncertainty is the fact that Bioquark has no preclinical evidence that its combination treatment is effective in animal models. The STAT piece details how the treatments have been tested individually for other conditions such as stroke and coma, but not in brain-dead patients. To further complicate things, there is no consensus on how to define brain death in patients, so patient improvements observed during the trial could be unrelated to the treatment.

STAT asked expert doctors in the field whether Bioquark’s strategy was feasible. Orthopedic surgeon Dr. Ed Cooper said that there’s no way electric stimulation would work, pointing out that the technique requires a functioning brain stem which brain-dead patients don’t have. Pediatric surgeon Dr. Charles Cox, who works on a stem cell treatment for traumatic brain injury and is unrelated to Bioquark, commented, “it’s not the absolute craziest thing I’ve ever heard, but I think the probability of that working is next to zero.”

But Pastor seems immune to the skepticism and naysayers.

“I give us a pretty good chance. I just think it’s a matter of putting it all together and getting the right people and the right minds on it.”

One scientist’s quest to understand autism using stem cells

April is National Autism Awareness Month and people and organizations around the world are raising awareness about a disorder that affects more than 20 million people globally. Autism affects early brain development and causes a wide spectrum of social, mental, physical and emotional symptoms that appear during childhood. Because the symptoms and their severity can vary extremely between people, scientists now use the classification of autism spectrum disorder (ASM).

Alysson Muotri UC San Diego

In celebration of Autism Awareness Month, we’re featuring an interview with a CIRM-funded scientist who is on the forefront of autism and ASD research. Dr. Alysson Muotri is a professor at UC San Diego and his lab is interested in unlocking the secrets to brain development by using molecular tools and stem cell models.

One of his main research projects is on autism. Scientists in his lab are using induced pluripotent stem cells (iPSCs) derived from individuals with ASD to model the disease in a dish. From these stem cell models, his team is identifying genes that are associated with ASD and potential drugs that could be used to treat this disorder. Ultimately, Dr. Muotri’s goal is to pave a path for the development of personalized therapies for people with ASD.

I reached out to Dr. Muotri to ask for an update on his Autism research. His responses are below.

Q: Can you briefly summarize your lab’s work on Autism Spectrum Disorders?

AM: As a neuroscientist studying autism, I was frustrated with the lack of a good experimental model to understand autism. All the previous models (animal, postmortem brain tissues, etc.) have serious experimental limitations. The inaccessibility of the human brain has blocked the progress of research on ASD for a long time. Cellular reprogramming allows us to transform easy-access cell types (such as skin, blood, dental pulp, etc.) into brain cells or even “mini-brains” in the lab. Because we can capture the entire genome of the person, we can recapitulate early stages of neurodevelopment of that same individual. This is crucial to study neurodevelopment disorders, such as ASD, because of the strong genetic factor underlying the pathology [the cause of a disease]. By comparing “mini-brains” between an ASD and neurotypical [non-ASD] groups, we can find anatomical and functional differences that might explain the clinical symptoms.

Q: What types of tools and models are you using to study ASD?

AM: Most of my lab takes advantage of reprogramming stem cells and genome editing techniques to generate 3D organoid models of ASD. We use the stem cells to create brain organoids, also called “mini-brains” in the lab. These mini-brains will develop from single cells and grow and mature in the same way as the fetal brain. Thus, we can learn about their structure and connectivity over time.

A cross section of a cerebral organoid or mini-brain courtesy of Alysson Muotri.

This new model brings something novel to the table: the ability to experimentally test specific hypotheses in a human background.  For example, we can ask if a specific genetic variant is causal for an autistic individual. Thus, we can edit the genome of that autistic individual, fixing target mutations in these mini-brains and check if now the fixed mini-brains will develop any abnormalities seen in ASD.

The ability to combine all these recent technologies to create a human experimental model of ASD in the lab is quite new and very exciting. As with any other model, there are limitations. For example, the mini-brains don’t have all the complexity and cell types seen in the developing human embryo/fetus. We also don’t know exactly if we are giving them the right and necessary environment (nutrients, growth factors, etc.) to mature. Nonetheless, the progress in this field is taking off quickly and it is all very promising.

Two mini-brains grown in a culture dish send out cellular extensions to connect with each other. Neurons are in green and astrocytes are in pink. Image courtesy of Dr. Muotri.

Q: We’ve previously written about your lab’s work on the Tooth Fairy Project and how you identified the TRPC6 gene. Can you share updates on this project and any new insights?

AM: The Tooth Fairy Project was designed to collect dental pulp cells from ASD and control individuals in a non-invasive fashion (no need for skin biopsy or to draw blood). We used social media to connect with families and engage them in our research. It was so successful we have now hundreds of cells in the lab. We use this material to reprogram into stem cells and to sequence their DNA.

One of the first ASD participants had a mutation in one copy of the TRPC6 gene, a novel ASD gene candidate. Everybody has two copies of this gene in the genome, but because of the mutation, this autistic kid has only one functional copy. Using stem cells, we re-created cortical neurons from that individual and confirmed that this mutation inhibits the formation of excitatory synapses (connections required to propagate information).

Interestingly, while studying TRPC6, we realized that a molecule found in Saint John’s Wort, hyperforin, could stimulate the functional TRPC6. Since the individual still has one functional TRPC6 gene copy, it seemed reasonable to test if hyperforin treatment could compensate the mutation on the other copy. It did. A treatment with hyperforin for only two weeks could revert the deficits on the neurons derived from that autistic boy. More exciting is the fact that the family agreed to incorporate St. John’s Wort on his diet. We have anecdotal evidence that this actually improved his social and emotional skills.

To me, this is the first example of personalized treatment for ASD, starting with genome sequencing, detecting potential causative genetic mutations, performing cellular modeling in the lab, and moving into clinic. I believe that there are many other autistic cases where this approach could be used to find better treatments, even with off the counter medications. To me, that is the greatest insight.

Watch Dr. Muotri’s Spotlight presentation about the Tooth Fairy Project and his work on autism.

Q: Is any of the research you are currently doing in autism moving towards clinical trials?

AM: IGF-1, or insulin growth factor-1, a drug we found promising for Rett syndrome and a subgroup of idiopathic [meaning its causes are spontaneous or unknown] ASD is now in clinical trials. Moreover, we just concluded a CIRM award on a large drug screening for ASD. The data is very promising, with several candidates. We have 14 drugs in the pipeline, some are repurposed drugs (initially designed for cancer, but might work for ASD). It will require additional pre-clinical studies before we start clinical trials.

Q: What do you think the future of diagnosis and treatment will be for patients with ASD?

AM: I am a big enthusiastic fan of personalized treatments for ASD. While we continue to search for a treatment that could help a large fraction of ASD people, we also recognized that some cases might be easier than others depending on their genetic profile. The idea of using stem cells to create “brain avatars” of ASD individuals in the lab is very exciting. We are also studying the possibility of using this approach as a future diagnostic tool for ASD. I can imagine every baby having their “brain avatar” analyses done in the lab, eventually pointing out “red flags” on the ones that failed to achieve neurodevelopment milestones. If we could capture these cases, way before the autism symptoms onset, we could initiate early treatments and therapies, increasing the chances for a better prognostic and clinical trajectory. None of these would be possible without stem cell research.

Q: What other types of research is your lab doing?

Mini-brains grown in a dish in Dr. Muotri’s lab.

AM: My lab is also using these human mini-brains to test the impact of environmental factors in neurodevelopment. By exposing the mini-brains to certain agents, such as pollution particles, household chemicals, cosmetics or agrotoxic products [pesticides], we can measure the concentration that is likely to induce brain abnormalities (defects in neuronal migration, synaptogenesis, etc.). This toxicological test can complement or substitute for other commonly used analyses, such as animal models, that are not very humane or predictive of human biology. A nice example from my lab was when we used this approach to confirm the detrimental effect of the Zika virus on brain development. Not only did we show causation between the circulating Brazilian Zika virus and microcephaly [a birth defect that causes an abnormally small head], but our data also pointed towards a potential mechanism (we showed that the virus kills neural progenitor cells, reducing the thickness of the cortical layers in the brain).

You can learn more about Dr. Muotri’s research on his lab’s website.


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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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