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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CIRM Grantees Reflect on Ten Years of iPS Cells

For the fourth entry for our “Ten Years of Induced Pluripotent Stem (iPS) Cells” series, which we’ve been posting all month, I reached out to three of our CIRM grantees to get their perspectives on the impact of iPSC technology on their research and the regenerative medicine field as a whole:

granteesStep back in time for us to August 2006 when the landmark Takahashi/Yamanaka Cell paper was published which described the successful reprogramming of adult skin cells into an embryonic stem cell-like state, a.k.a. induced pluripotent stem (iPS) cells. What do you remember about your initial reactions to the study?

Sheng Ding, MD, PhD
Senior Investigator, Gladstone Institute of Cardiovascular Disease
Shinya had talked about the (incomplete) iPS cell work well before his 2006 publication in several occasions, so seeing the paper was not a total surprise.

Alysson Muotri, PhD
Associate Professor, UCSD Dept. of Pediatrics/Cellular & Molecular Medicine
At that time, I was a postdoc. I was in a meeting when Shinya first presented his findings. I think he did not give the identity of the 4 factors at that time. I was very excited but remember hearing rumors in the corridors saying the data was too good to be true. Soon after, the publication come out and it was a lot of fun reading it.

Joseph Wu, MD, PhD
Director, Stanford Cardiovascular Institute
I remember walking to the parking lot after work. One of my colleagues called me on my cell phone and he asked if I had seen “the Cell paper” published earlier that day. I said I haven’t and I would look it up when I get back home. I read it that night and found it quite interesting because the concept was simple but yet powerful.

How soon after the publication did you start using the iPSC technique in your own research? At that time, what research questions were you able to start exploring that weren’t possible in the “pre-iPS” era?

I think many of us in the (pluripotent stem cell) field quickly jumped on this seminal discovery and started working on the iPSC technology itself as, at the time, there were many aspects of the discovery that would need to be better understood and further improved for its applications.

Immediately after the first mouse Cell paper, but I started with human cells. There were some concerns if the 4 factors will also work in humans. Nonetheless, I start using the mouse cDNA factors in human cells and it worked! I was amazed to witness the transformation and see the iPSC colonies in my dish – I showed the results to everyone in the lab.

Soon after, the papers showing that the procedure worked in human cells were published but I already knew that. Thus, I started to apply this to model disease, my main focus. In 2010, we published the modeling of the first neurodevelopmental disease using the iPSC technology. It is still a landmark publication, and I am very happy to be among the pioneers who believed in the Yamanaka technology.

We started working on iPS cells about a couple of months after the initial publication. To our surprise, it was incredibly easy to reproduce, and we were able to get successful clones after a few initial attempts, in part because we had already been working on human embryonic stem (ES) cells for several years.

I think the biggest advantage of iPS cells is that we can know the medical record of the donor. So we can study the correlation between the donor’s underlying genetic makeup and their resulting cellular and whole-body characteristics using iPS cells as a platform for integrating these analyses. Examining these correlations is simply not possible with ES cells since no adult donor exists.

Dr. Ding, what do you think made you and your research team especially skilled at pioneering the use of small molecules to replace the “Yamanaka” reprogramming factors?

We had been working on identifying and using small molecules to modulate stem cell fate (including cell proliferation, differentiation, and reprogramming) before iPS cell technology was reported. So when the iPS cell work was reported, it was obvious to us that we could apply our expertise in small molecule discovery to better understand and improve iPS cell reprogramming and replace the genetic factors by pharmacological approaches.

Now, come back to the present and reflect on how the paper has impacted your research over the past 10 years. Describe some of the key findings your lab has made over the past 10 years through iPSC studies

We’ve worked on three aspects that are related to iPS cell research: one is to identify small molecule drugs that can functionally replace the genetic reprogramming factors, and enhance reprogramming efficiency and iPS cell quality (to mitigate risks associated with genetic manipulation, to make the iPS cell generation process more robust and efficient, and reduce the cost etc).

Second is to better understand the reprogramming mechanisms, that would allow us to improve reprogramming and better utilize cellular reprogramming technology. For example, we had uncovered and characterized several fundamental mechanisms underlying the reprogramming process.

The third is to “repurpose/re-direct” the iPS cell reprogramming into directly generating tissue/organ-specific precursor cells without generating iPS cell (itself, which is tumorigenic and needs to be differentiated for most of its applications). This so-called “Cell-Activation and Signaling-Directed/CASD” reprogramming approach allowed us to directly generate cells in the brain, heart, pancreas, liver, and blood vessels.

My lab has focused on the use of iPS cells to model autism spectrum disorder, a condition that is very heterogeneous both clinically and genetically. Previous models for autism, such as animals and postmortem tissues, were limited because we could not have access to live neurons to test experimentally several hypotheses. Thus, the attractiveness of the iPS cell model, by capturing the genome of patients in pluripotent stem cells and then guide them to become neural networks.

While the modeling in a dish was a great potential, there were some clear limitations too: the variability in the system was too high for example. My lab has worked hard to develop a chemically-defined culture media (iDEAL) to grow iPS cells and reduce the variability in the system. Moreover, we have developed robust protocols to analyze the morphology and electrophysiological properties of cortical neurons derived from iPS cells. We have used these methods to learn more about how genes impact neuronal networks and to screen drugs for several diseases.

We also used these methods to create cerebral organoids or “mini-brains” in a dish and have applied this technology to test the impact of several genetic and environmental factors. For example, we recently showed that the Zika virus could target neural progenitor cells in these organoids, leading to defects in the human developing cortex. Without this technology, we would be limited to mouse models that do not recapitulate the microcephaly of the babies born in Brazil.

Our lab has taken advantage of the iPS cell platform to better understand cardiovascular diseases and to advance the precision medicine initiative. For example, we have used iPS cells to elucidate the molecular mechanisms of diseases related to an enlarged heart, cardiac arrhythmias, viral- and chemotherapy-induced heart disease, the genetics of coronary artery disease, among other diseases. We have also used iPS cells for testing the safety and efficacy of various cardiovascular drugs (i.e., “clinical trial in a dish”).

How are your findings important in terms of accelerating stem cell treatments to patients with unmet medical needs?

Better understanding the reprogramming process and developing small molecule drugs for enhancing reprogramming would allow more effective generation of safe stem cells with reduced cost for treating diseases or doing research.

We work with two concepts. First, we screen drugs that could repair the disorder at a cellular level in a dish, hoping these drugs will be useful for a large fraction of autistic individuals. This approach can also be used to stratify the autistic population, finding subgroups that are more responsive to a particular drug. This strategy should help future clinical trials.

In parallel, we also work with the idea of personalized medicine by using patient-derived cells to create “disease in a dish” models in the lab. We then examine the genomic information of these cells to help us find drugs that are more specific to that individual. This approach should allow us to better design the treatment, testing ideal drugs and dosage, before prescribing it to the patient.

The iPS cell technology provides us with an unprecedented glimpse into cardiovascular developmental biology. With this knowledge, we should be able to better understand how cardiac and vascular cells regenerate in the heart during different phases of human life and also during times of stress such as in the case of a heart attack. However, to be able to translate this knowledge into clinical care for patients will take a significant amount of time. This is because we still need to tackle the issues of immunogenicity, tumorigenicity, and safety for products that are derived from ES and iPS cells. Equally importantly, we need to understand how transplanted cells integrate into the patient because based on our experience so far, most of the injected cells die upon transplant into the heart. Finally, the economics of this type of personalized regenerative medicine is a daunting challenge.

Finally, it’s foolhardy to predict the future but, just for fun, imagine that I revisit you in August 2026. What key iPSC-related accomplishments do you think your lab will achieve by then?

We are hoping to have cell-based therapy and small molecule drugs developed based on iPS cell-related research for treating human diseases. Particularly, we are also hoping our cellular reprogramming research would lead us to identify and develop small molecule drugs that control tissue/organ regeneration in vivo [in an animal].

We hope to have improved several steps on the neural differentiation, dramatically reducing costs and increasing efficiency.

We would like to use the iPS cell platform to discover several new drugs (or repurpose existing drugs) for our cardiovascular patients; to replace the current industry standard of drug toxicity testing using the hERG assay (which I believe is outdated); to predict what medications patients should be taking (i.e., precision cardiovascular medicine); and to elucidate risk index of genetic variants (in combination with genome editing approach).

What’s the big idea? Or in this case, what’s the 19 big ideas?

supermarket magazineHave you ever stood in line in a supermarket checkout line and browsed through the magazines stacked conveniently at eye level? (of course you have, we all have). They are always filled with attention-grabbing headlines like “5 Ways to a Slimmer You by Christmas” or “Ten Tips for Rock Hard Abs” (that one doesn’t work by the way).

So with those headlines in mind I was tempted to headline our latest Board meeting as: “19 Big Stem Cell Ideas That Could Change Your Life!”. And in truth, some of them might.

The Board voted to invest more than $4 million in funding for 19 big ideas as part of CIRM’s Discovery Inception program. The goal of Inception is to provide seed funding for great, early-stage ideas that may impact the field of human stem cell research but need a little support to test if they work. If they do work out, the money will also enable the researchers to gather the data they’ll need to apply for larger funding opportunities, from CIRM and other institutions, in the future

The applicants were told they didn’t have to have any data to support their belief that the idea would work, but they did have to have a strong scientific rational for why it might

As our President and CEO Randy Mills said in a news release, this is a program that encourages innovative ideas.

Randy Mills, Stem Cell Agency President & CEO

Randy Mills, CIRM President & CEO

“This is a program supporting early stage ideas that have the potential to be ground breaking. We asked scientists to pitch us their best new ideas, things they want to test but that are hard to get funding for. We know not all of these will pan out, but those that do succeed have the potential to advance our understanding of stem cells and hopefully lead to treatments in the future.”

So what are some of these “big” ideas? (Here’s where you can find the full list of those approved for funding and descriptions of what they involve). But here are some highlights.

Alysson Muotri at UC San Diego has identified some anti-retroviral drugs – already approved by the Food and Drug Administration (FDA) – that could help stop inflammation in the brain. This kind of inflammation is an important component in several diseases such as Alzheimer’s, autism, Parkinson’s, Lupus and Multiple Sclerosis. Alysson wants to find out why and how these drugs helps reduce inflammation and how it works. If he is successful it is possible that patients suffering from brain inflammation could immediately benefit from some already available anti-retroviral drugs.

Stanley Carmichael at UC Los Angeles wants to use induced pluripotent stem (iPS) cells – these are adult cells that have been genetically re-programmed so they are capable of becoming any cell in the body – to see if they can help repair the damage caused by a stroke. With stroke the leading cause of adult disability in the US, there is clearly a big need for this kind of big idea.

Holger Willenbring at UC San Francisco wants to use stem cells to create a kind of mini liver, one that can help patients whose own liver is being destroyed by disease. The mini livers could, theoretically, help stabilize a person’s own liver function until a transplant donor becomes available or even help them avoid the need for liver transplantation in the first place. Considering that every year, one in five patients on the US transplant waiting list will die or become too sick for transplantation, this kind of research could have enormous life-saving implications.

We know not all of these ideas will work out. But all of them will help deepen our understanding of how stem cells work and what they can, and can’t, do. Even the best ideas start out small. Our funding gives them a chance to become something truly big.

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