February 19, 2016
Breaking Through Barriers
A new approach to autism research offers hope to families and spares animal lives
Jen Bidlovski would give anything to break through the silence that surrounds her son Milo. Diagnosed with autism as a young child, he spoke the word “up” at age 1, then never talked again. Milo is now 12. Locked in a small, interior world, even his most urgent needs—relief from the severe pain of a recent attack of appendicitis—remain unheard. Bidlovski has sacrificed her marriage, a music career, a house and most of her savings in search of a way to free him. Today, she’s a single mom living with Milo and a younger son in an apartment in San Diego. She has one hope left. It rests on something small but potentially very powerful: a single baby tooth.
It was the third of Milo’s baby teeth that she rushed to the office of researcher Alysson Muotri at the University of California, San Diego. Unlike the first two, the pulp was still fresh enough that it could be used to pioneer a new type of autism research. Instead of examining the brains of mice, which for more than 60 years lab workers across the country have raised, decapitated and autopsied, Muotri is examining human cells grown from dental pulp. They have been donated by Milo and 300 other children, some autistic, some not.
Until she stumbled upon Muotri’s research online in 2010, Bidlovski had little expectation of reaching Milo. At home, he sits for hours listening to classical music on his headphones, staring into an iPad and playing with an ever-present strand of beads. He is there in the room with her, but he is not really present. Bidlovski has tried almost all of the available treatments: gluten-free and dairy-free diets, vitamins, behavioral approaches. She saw some of these work for other children. But Milo remains nonverbal. The days he spends in school bring no progress.
“It’s kind of like a mini death everyday,” Bidlovski says. “No friends. No toys. And you live with it every day. It just won’t end.”
Scientists have come to realize that there is no one cause of autism—no one missing or defective gene behind the problem. Instead, there are a variety of causes. There is also no one “autism.” Individuals with the disorder show varying degrees of difficulty communicating and making social connections, and varying degrees of repetitive behaviors. They inhabit different places along a spectrum that ranges from mild to severe.
The complexity of the disorder means looking at the brains of mice genetically modified to show some symptoms mimicking autism, as many researchers still do, is unlikely to reveal how to treat what is happening in the brains of humans with autism in general, let alone in Milo’s brain. But Muotri’s research might well deliver this kind of personalized medicine. That’s because his lab is looking at actual human brain cells. In Milo’s case, they are miniature, greatly simplified models of his individual brain, grown from baby tooth pulp cells that contain his unique DNA.
By examining brain cells grown from the baby teeth, Muotri has found one common characteristic that he believes contributes to different types of autism: autistic brain cells have many fewer branches and connections called synapses than brain cells from neurotypical people: They are less able to communicate with one another. In September, brain cells derived from baby teeth from Muotri’s lab were packed into a cooler and flown across the country to the National Institutes of Health in Bethesda, Maryland. By then the cells had grown into networks of neurons that rested in rows of little wells, 384 to a plate. In a process known as high-throughput testing, NIH robots—giant hydraulic arms—swiftly moved the plates at timed intervals to expose the cells to more than 55,000 potential drugs. These tests will determine which, if any, of the chemicals might stimulate the cells to grow more connections and function more typically.
This month or next, Muotri may know what might have taken scientists in previous generations lifetimes to discover. No mice will die. And parents such as Bidlovski may finally get effective treatments for their children. “Think of all these kids stuck in their brains—my kid’s wonderful, but he is trapped,” says Bidlovski. “There’s nothing that could be more important to me than to hear my son’s voice.”
Muotri is among a growing number of researchers who have abandoned or greatly reduced animal testing in favor of more effective human-biology based approaches. In many cases, researchers, like Muotri, have adopted nonanimal methods because they work better. “I want the best model to learn about the human brain,” Muotri says. “And the best model is this.”
In other cases, scientists are adopting nonanimal methods for ethical reasons. Elan Ohayon, who collaborates with Muotri on the baby tooth research, works with his wife, Ann Lam, at Green Neuroscience Laboratory, which the couple created as an alternative to labs that harm animals, human health and the environment. They call their research “human-based.”
“People will say, ‘It’s great to do animal research; you have to compromise.’ And we’re saying, ‘No,’ ” says Ohayon.
What was once seen as inevitable—the use of tens of millions of animals in research labs—is slowly decreasing, with the encouragement of The HSUS, Humane Society International (HSI) and the scientists themselves. In the U.S., students earning their doctorates in the biological sciences continue to be required to raise and then kill mice in a grim rite of passage. But now some students are also being trained to employ nonanimal techniques.
Around the world, scientists are using sophisticated, nonanimal approaches to tackle research problems they haven’t been able to solve with experiments on mice or other animals. Dutch researcher Anja van de Stolpe is using “organ on a chip” technology to better understand and treat autoimmune disorders. She grows human blood vessel tissue and then exposes it to lymph node and other immune system tissue. German researcher Fozia Noor is taking a similar approach to cholestatic liver disease, which cuts off the flow of bile to the gall bladder.
Mice are not mini humans. Our brain is way more complex, and our metabolism and immune system are very different.”
- Researcher Alysson Muotri
In December, HSI brought van de Stolpe, Noor and Muotri together with European Union officials, funding bodies and other stakeholders at a workshop in Brussels for the launch of the BioMed21 Initiative (short for "biomedical research for the 21st century," a project co-financed by World Animal Protection). The three scientists joined independent experts and more researchers doing human-based work on Alzheimer’s, autism, autoimmune disorders, asthma and liver disease. The message delivered: The animal model is obsolete.
HSI is organizing similar gatherings in Brazil, South Korea, India, Japan and also in the U.S., working with The HSUS and federal agencies, says Troy Seidle, HSI director of research and technology. The hope is that a significant portion of the $30 billion NIH and its equivalents provide for medical research each year can be directed to nonanimal models.
“There’s such tremendous potential for nonanimal research, but there’s no road map for scientists or research funders to follow,” Seidle says. “HSI and The HSUS are at the forefront of working with the biomedical community to develop that road map. We’re building this step-by-step global discussion.”
Scientists are ready. A paper on nonanimal approaches to Alzheimer’s research written by Gillian Langley, senior science adviser to HSI, has been downloaded more than 2,000 times. It details the limitations of using genetically modified mice, which develop Alzheimer’s-like conditions but nothing close enough to the actual human disease to allow scientists to develop effective treatments.
One of the world’s foremost Alzheimer’s researchers, Rudolph Tanzi of Harvard University, worked with mice for 20 years but has switched to “Alzheimer’s in a dish” experiments with lab-grown human neurons. He says he can test drugs 10 times faster and 10 times more cheaply—with far more meaningful results. Already, he has identified 12 drugs the FDA approved for other conditions that could treat Alzheimer’s. One is going into clinical trials. “The animal models did not fully repeat the pathology,” he says. “You could get pieces of the pathology, but you could not get the disease.”
No one is officially declaring an end to animal research. Discoveries made by nonanimal methods are still greeted with suspicion—and demands that they be reproduced using animals—while funding agencies and peer reviewers still require “proof” from animal studies. However, a shift is underway, Langley says, especially after repeated failures of drugs developed through animal experimentation. “If you probe into the scientific community and ask them why they use animals, they’re always defensive,” she says. “But if you’re at a meeting, they’re more open. There’s a lot of concern that drug development is becoming very costly and failing. People are looking for answers.”
Early in his career, Muotri took the lives of a lot of mice. He thought he had to. He was studying Rett Syndrome, a neurological disorder with symptoms similar to autism. He followed procedures that are standard in most U.S. labs. As mice developed a genetically-induced model of the disorder, Muotri would kill them to study their brains—the number of synapses and the circuitry. “I never liked it—it was never pleasant,” he says. “But I felt that that was the only way we could help the families. All the time when I had to kill, I was saying in my mind, ‘I’m going to kill you, but this is for a good thing.’”
Muotri’s work on mice led to several discoveries, which may or may not apply to humans. His big break, though, has come through studies of lab-cultured human cells, like the ones he is growing for autism. Years after the mice research, the stunted branches of neurons grown from the DNA of someone with Rett Syndrome responded to a substance called insulin growth factor. The neurons grew more branches. Muotri published a paper in 2010.
The technology that made all this possible was revealed to Muotri at a 2006 conference by Dr. Shinya Yamanaka. Within six years, the Japanese scientist would be awarded a Nobel Prize. But at the time there were not many people in the room, and there were no more than 100 in the world in the field of stem cell research. That would all change. Because Yamanaka had found a way to take adult cells that have developed into, say, tooth pulp or skin or hair or blood, and revert them back to “pluripotent” stem cells that have the potential to become any cell. All it required, Yamanaka explained, was exposing the adult cells to four specific genes.
Half of the members of the small audience were skeptical. But the other half, including Muotri, were excited: “I immediately thought, ‘Wow, I could use that!’”
Until the conference, Muotri had only two ways to see what was happening in autistic brains: He could use the donated brains of people with autism who had died—but there are only 30 to 50 such brains in the world—or he could use mice genetically modified to display autism-like symptoms, such as remaining in the corners of their cages when other mice are introduced.
Muotri didn’t think testing drugs on mice would lead anywhere. “Mice are not mini humans,” he says. “Our brain is way more complex, and our metabolism and immune systems are very different.”
You know this thing is living and it troubles you. Either you dissociate or you go home and feel horrible.”
- Researcher Elan Ohayon
Yamanaka’s technique allowed Muotri to take discarded cells from people with autism and turn them into live brain cells. Figuring that the easiest, least threatening way to gather cells was through baby teeth, Muotri formed a network that grew to 3,500 families in California, Brazil and Europe. Viable pulp cells were transformed into neurons, then grown into neuronal networks that can survive for up to six months.
Muotri works at a UCSD research center, clustered with other high-tech facilities—the Scripps Research Institute, the Salk Institute for Biological Studies. To the west, you can see the Pacific, or rather the place where the land drops to the breaking waves below. There’s a spacious terrace with young women in yoga pants and retirees lunching on patio furniture under umbrellas. Beyond lies a vast lobby with gleaming floors.
Muotri’s office is a glass cube perched on the side of the building overlooking the ocean. On one wall hang framed covers of stories about his work in the journal Cell and in Scientific American magazine. Behind his desk is a smaller frame containing something more important: a picture of his son, Ivan. Like Milo, Ivan is severely autistic. He is 9, born around the time that Muotri learned it was possible to revert adult cells to stem cells. When Muotri adopted his son, the researcher was already studying autism. Ivan, who is part of the baby tooth study, is a constant motivation as he looks for treatments.
“Only those who live with the condition know how hard daily life is,” Muotri says. “[Ivan] is such a fun and energetic boy. I would be happy if he could become an independent person.”
On a shelf in Muotri’s office is a display of models of human and near-human skulls: human ancestors and primates. Muotri has long been fascinated with the evolution of the human brain. Chimpanzee brains, he explains, are wired so that the animals can deal with up to 50 other individuals at once. That’s about as large as chimp colonies can get. Typical human brains, by contrast, are wired so that people can deal with three times as many individuals. That requires billions and billions of neurons. And trillions and trillions of synapses—the connections that people with autism lack.
“The biggest thing that makes us human is social interaction,” says Muotri. “The ability to communicate with other people.”
In order to understand how autism limits this ability, Muotri takes the neuronal networks grown from baby teeth and measures the electrical activity in these “mini brains.” He does this by putting them on microchips and using embedded electrodes to detect neurons firing. The results are visible on a computer screen, just as they would be on an EEG or electroencephalogram: Patterns in jagged lines of red, yellow, green and blue erupt. Muotri sends the data to Ohayon.
In the big lab area shared by Muotri’s team and other groups, his graduate and post-doctoral students care for cells. They must visit the lab six to seven days a week during the six to eight weeks it takes to grow a neuron. They “feed” the cells liquids that help them grow and divide. Brain cells take as much attention as mice.
Like Muotri, postdoctoral student Pinar Mesci has taken the lives of a lot of mice. As a graduate student in France, she raised mice genetically modified to develop Lou Gehrig’s disease. Some were given a potential treatment. All were killed and autopsied. For those who developed the disease, it was a mercy—they were euthanized to spare them the suffering of the end stages, which includes paralysis. Mesci would give mice in the study a drug to put them in a coma. Then she would decapitate them or kill them with carbon dioxide. She did it because the research carried the possibility that humans might be spared the agony of the disease. “The thing that you have to do is not to make the mouse suffer,” she says. “It has to be fast. It has to be painless.”
Mesci came to Muotri’s lab to learn his induced stem cell technique. She’s growing stem cells into neurons and also brain cells called microglia, which are part of the immune system and help make connections in the developing brain. Mesci wants to investigate whether substances secreted by microglia could treat Rett Syndrome. She is testing the effect of the substances on neurons.
Mesci goes to a small room off the main lab, where her cells are stored in an incubator. One set of neurons is just a day old. Under a digital microscope, the cells are scattered blobs with thin projections reaching out toward one another, like spindly arms. A second set is one month, 19 days old. These cells are organizing themselves, forming spheres of clusters of neurons.
When the cells are developed enough, Mesci exposes the neuronal networks to the substances produced by the microglia. After 24 hours, she fixes the cells—or stops their growth—so she can look at them under the microscope. In past days, when she worked with mice, this step in her work required decapitating the animals so she could see what was happening in their brains. These days, it only requires exposing the cells, growing in rows of wells, to 40 percent formaldehyde. Technically, it’s the same as killing the cells, but there’s a big difference between cutting off an animal’s head and methodically going down rows with droppers to administer a chemical.
Beatriz Freitas, another postdoctoral student in the lab, says this difference is why she quit a lab at Harvard and came to Muotri’s, abandoning her former research project. She says she originally got into biology because she wanted to save the tigers. She found herself working with mice, considered expendable. But she couldn’t discount the lives of animals, even small ones.
Across town, hidden along a strip of shopping centers and car dealerships, is the Green Neuroscience Laboratory, where Muotri is sending EEG data from the lab-grown neuronal networks for Ohayon to analyze. Though Ohayon and Lam were both postdoctoral students at the Salk Institute, everything about their own much smaller lab challenges prevailing practices.
The building materials are reclaimed. Much of the power comes from solar panels on the roof of the building, which is shared with other start-ups. There are plants and an environment free from toxic chemicals or research animals carrying diseases. It’s safe enough that the couple brings their 2-year-old daughter, Gali, to visit. Lam and Ohayon use open-source software and hardware and do not believe in patents or proprietary science.
The couple created the lab to express their own ethics. And also to show other scientists-in-training what is possible. “When they see it as an impossibility, it’s a failure of empathy. It’s a failure of imagination,” Ohayon says.
As a graduate student, Lam was required to learn two different ways to kill lab animals. “It was very hard,” she says. “Everyone was very quiet. The idea is to create a detached, technical environment.”
Neuroscience students are commonly taught to deeply anesthetize animals, pump them full of formaldehyde to fix their brains and then decapitate them with a guillotine. “The body will move,” Ohayon says. “They’re not supposed to feel, but we’re not really sure. ... You know this thing is living and it troubles you. And it’s always there—either you dissociate, or you go home and feel horrible.”
Lam studies epilepsy without the use of animals by examining parts of human brains removed during surgery to reduce seizures. She starts with pieces of tissue about the size of a fingernail on a person’s pinky. Then she slices this tissue into thin sections to make images that she can stain and magnify. Several times a year, she travels with these to Stanford University’s synchrotron, which is a city block in size. This huge circular particle accelerator produces powerful X-rays that have been used to reveal the composition of the feathers of the birdlike dinosaur archaeopteryx. Lam is looking for concentrations of metals, like zinc and iron, in the brain tissue. Such metals can work like neurotransmitters, allowing different cells to talk to one another. Too much or too little of them can cause disease. Lam believes this may play a role in epilepsy, and so she is creating an “atlas” of where they lie in the brain.
Ohayon builds computer models of the neuronal networks—”neurocomputational simulations”—and sees what electrical activity they generate. He’s hoping this will give him insight into how the brain evolved, into attention and memory, and into diseases such as epilepsy and Alzheimer’s. If he were working with animals, in order to do this type of research, he would have to “spike” their brains, inserting electrodes into the living tissue. With his simulations, he decides on the number of connections between cells and then watches the computer screen: What look like jagged mountain peaks explode upward in scattered places to varying heights, die away, and then reappear in different spots across a three-dimensional square.
Ohayon is helped by a simple robot that a graduate student in the lab built for less than $200. When he plugs the robot into the computer, the simulated brain activity is transferred into real-world robot movements. If the simulated networks have few connections, the activity and the movement stop. It’s as if the “brain” had a seizure or went into a coma. If there are many connections, the activity continues.
Ohayon is studying the electrical activity measurements from Muotri’s lab-grown neuronal networks, trying to create mathematical models that will explain differences in the patterns from autistic and nonautistic cells. He wants to discover how the amount and speed of the electrical signals relate to the number and type of connections between neurons. The hope is that this will provide insight into what is going wrong in the brains of people with autism. That knowledge could help researchers develop treatments.
If autism research still focused solely on mice, Bidlovski would have no expectation of anything more. There could be no discovery that might help her son talk. But now that science has allowed better and more humane research, she is daring to hope: for a part of her son she feels was stolen to be returned to her, for the chance to one day know what he is thinking, to reach through the barriers and finally communicate with her son. For that one word.
“I’d give anything to just hear him talk,” she says. “I’d give anything to just hear him say, ‘Mom.’ ”
Changing the Face of Cosmetics Testing
Just as new technologies are allowing more research scientists to abandon animal studies, they are also freeing cosmetic companies from the need to test their products on animals. In 2015, a San Diego company called Organovo announced it will be manufacturing skin-like tissue from human cells for the cosmetics giant L’Oreal. Organovo will be using 3-D bioprinting to create a living tissue that mimics the form and function of actual human skin.
“The potential for where this new field will take us is boundless,” says Guive Balooch, global vice president of L’Oreal’s technology incubator, in a press release.
Organovo will sell the skin exclusively to L’Oreal, which until recently used reconstructed skin tissue produced at a company research center to test the safety and effectiveness of its products. The Organovo product is expected to be much faster to produce than the tissue L’Oreal now uses, which is cultured from skin cells donated after plastic surgeries.
In the same way regular 3-D printers are programmed to build objects layer by layer out of plastic, 3-D bioprinting machines are programmed to build tissue layer by layer out of cells that have particular structures or patterns. Afterward, the cells organize and fuse to become living tissue.
Michael Renard, Organovo’s executive vice president of commercial operations, says the technology—which has also been used to produce liver and kidney tissue—may not mean the end of all animal testing, but it will allow companies to reduce animal use.
“We’re in the business of building human tissue,” he says. “Animals are expensive, and in some cases [the findings] are not all that accurate.”