Thursday, June 21, 2018

By Laura Hedli

There’s a really good chance the whole thing might never have happened as it did. Sergiu Pasca, MD, then a recent medical graduate from Transylvania, Romania, had to convince Ricardo Dolmetsch, PhD, that he was able to excel as a neuroscience postdoctoral scholar at one of the top academic research institutions in the U.S. Add to this already formidable challenge that Dr. Pasca had no PhD, no funding, very little formal bench training, and was still living in Romania where he had only recently started learning English.

Meanwhile, Dr. Dolmetsch, then an associate professor of neurobiology, was perilously close to running out of money for his lab. He had decided to change its focus, trading work on calcium channels for the study of autism using a new method straight out of a science fiction movie—funders and academicians weren’t buying it. They didn’t understand his vision and weren’t willing to take a risk on a basic researcher who had no experience in psychiatry.

Both Drs. Dolmetsch and Pasca persisted despite discouragement; the former received a high-risk, high-reward NIH award to keep the lights on, and the latter secured a small grant, enough to get Dr. Dolmetsch to take a chance on him. Even though Dr. Pasca was inexperienced, Dr. Dolmetsch found him to be smart, enthusiastic, and, as an added bonus, he had experience with children who had been diagnosed with mental disorders.

Once stateside, Dr. Pasca began work with his new mentor to make human cell models of autism spectrum disorders. The first step in this process involved turning back the clocks on development, reprogramming skin cells (taken from living humans) back down into stem cells. Then, they coaxed those stem cells to become neurons in a petri dish. If this this two-way metamorphosis sounds extremely difficult to pull off, that’s because it was.

Grants from Stanford Child Health Research Institute (CHRI) buoyed the researchers at this critical juncture. As Dr. Dolmetsch explains, “You can't run a deficit at Stanford, right? There wasn't a huge amount of sympathy for this guy who worked a lot but couldn't seem to raise any money. It [CHRI] actually stopped the lab from closing.”

Fewer than 2 percent of MDs conduct research in the U.S. It was amazing that CHRI thought very early on that MDs who want to do research should have a separate funding track.

Dr. Dolmetsch was named a Barbara and John Packard Faculty Scholar in 2011 to study the underlying basis of autism spectrum disorders using what’s called pluripotent stem cell (iPSC) technology. (That’s the fancy term for the cellular metamorphosis.) Faculty Scholars receive $500,000 over five years, though Dr. Dolmetsch had to return a portion of these funds back to CHRI when he left to pursue his current position as Global Head of Neuroscience at Novartis in Cambridge, Massachusetts.

Dr. Pasca was named Tashia and John Morgridge Endowed Postdoctoral Fellow in Pediatric Translational Medicine, receiving $120,000 that supported his postdoctoral work in Dr. Doltmetsch’s lab for two years (2009-2011) and helped launch his career as a promising young investigator.

“Fewer than 2 percent of MDs conduct research in the U.S. It was amazing that CHRI thought very early on that MDs who want to do research should have a separate funding track,” Dr. Pasca says of CHRI’s decision to make two distinct award tracks for MDs and PhDs. He notes that PhDs will often have a more impressive publication track record than MDs who are coming out of medical school and want to pursue basic science yet have been busy with clinical training.

How to unspill water

The Dolmetsch lab aimed to create models of neurons derived from the skin of human patients in order to study psychiatric disorders. While this methodology has quickly gained traction, 10 years ago, the idea of applying molecular biology to answer psychiatric questions was novel. How would one get access to a living person’s brain tissue without taking a biopsy?

“In psychiatry, we suffer from an ‘oncology envy syndrome’ because we see how fast oncology has been moving for the last few decades towards personalized cures and treatment,” Dr. Pasca says. Tumors are resected and studied, and doctors and patients accept (perhaps even expect) treatment to be more aggressive. Dr. Pasca’s work is borne out of a frustration with a modus operandi that is outdated and too imprecise in an age where we’re moving more and more toward specificity. “In psychiatry, we diagnose in the way we were doing 150 years ago. We treat disorders with a few drugs that were discovered by chance without really trying to address the core fundamental issues pathophysiologically.”

Dr. Pasca began in the Dolmetsch lab with a longstanding interest in investigating autism. In medical school, he had studied metabolism and the role of various enzymes in the blood of children with autism spectrum disorders. Around the same time, Dr. Dolmetsch, who was known for his work on calcium channels, became interested in trying to understand the underlying cellular and molecular basis of neurodevelopmental disease after his oldest son was diagnosed with autism. His personal experience led him to switch gears in his lab. But there was a problem—all his existing funding was designated for research that had nothing to do with the work his lab was now pursuing.

Dr. Dolmetsch was able to initially pursue this new direction and bring on Dr. Pasca prior to CHRI funding thanks to an NIH Pioneer Award he received in 2008. The award supports exceptionally creative scientists proposing pioneering approaches to major challenges in biomedical and behavioral research. Dr. Pasca secured a tiny grant from International Brain Research Organization in order to come to California from Romania. The researchers agreed to work together on iPSC technology even though their ideas ran seemingly counter to general biologic understanding.

The basic idea was that development is a kind of one-directional arrow. It's like spilling water; you spill water, you don't unspill water, right?

“The basic idea was that development is a kind of one-directional arrow,” Dr. Dolmetsch says. “It's like spilling water; you spill water, you don't unspill water, right?”

But as it turns out, you can, and the researchers did. They started having some success in putting water back into the glass, so to say.

Before figuring out how to create neurons, another postdoctoral scholar in the lab, Masayuki Yazawa, PhD, had generated cardiomyocytes using iPSC technology and skin cells from both healthy children and children with a heart condition. It was an easier challenge. In fetal development, it takes 26 weeks for the cortex to form, whereas the heart beats within 21 days. “When we made the little human hearts, it was amazing—they beat beautifully,” Dr. Dolmetsch recalls. “They beat sort of like human hearts at 60 beats per minute. And the cells from the kids with the disease, their hearts beat way more slowly.”

A breakthrough in the lab came in 2011 when Dr. Pasca developed neuron cultures from skin cells and identified phenotypic differences between diseased and control cells. It was one of the first models of neuropsychiatric disorders in a dish and was reported as a cover story in Nature Medicine.

Following an unconventional postdoctoral trajectory, Dr. Pasca wouldn’t have been able to stay on in the Dolmetsh lab after 2011 without funding. His work during his years as a CHRI Morgridge Fellow helped him gain credibility and recognition as a scientist. He received a NARSAD Young Investigator Grant in 2012, and the ensuing years in the lab fostered his relationship with his mentor.  

“Ricardo and I clicked so well,” Dr. Pasca says, noting that their goals have been aligned since the beginning of their time together. “We had late night conversations about the major questions in the field and how to approach them experimentally. He's been just an amazing mentor.”

The promise of molecular psychiatry

In their work on iPSC technology, the researchers often studied cells of a person with Timothy Syndrome, a disorder that affects the heart, digits, and nervous system and impacts only a few dozen people worldwide according to the National Institutes of Health Genetics Home Reference. In order to get the samples, the researchers contacted the person who first described the syndrome and who knows those affected. Individuals who wanted to participate flew to Stanford’s campus, where they received a skin biopsy.

The rarity of Timothy Syndrome paired with success of the research is notable. New iPSC technology may significantly impact the field of precision medicine, as an individual’s own skin cells are used to understand their unique pathology and response to treatment.

“Because your DNA is more or less the same in all the cells in your body, you can make neurons with the same DNA by starting with any cell in your body, even a skin cell,” Dr. Dolmetsch says.

After five years being a postdoctoral scholar in Dr. Dolmetsch’s lab, Dr. Pasca started his own lab at Stanford within the Department of Psychiatry and Behavioral Sciences. He and his wife Anca Pasca, MD, currently a Stanford neonatal-perinatal medicine fellow, discovered how to create what he now calls brain-region-specific three-dimensional cultures. To do this they coaxed the cells to form into a ball shape and self-organize so as to function as a proxy of the human cortex. (A winter 2018 feature story in Stanford Medicine magazine affectionately called these clumps of cells brain balls.) A Nature Methods article in 2015 chronicled the approach to creating the 3D cultures—Anca was the first author and Sergiu the senior author.

“It's not the entire brain—that's not the purpose,” Dr. Pasca says of the 3D cultures. “The purpose is really to try to capture aspect of human function and development that we did not previously have access to.” The technology the Pasca lab has been developing has allowed the cultures, now also known as brain organoids, to model more of the complexity of the human brain and stay viable for longer periods of time—up to 800 days. In fact, after about a year, the glial cells within brain organoids show a postnatal signature. As documented in an article in Neuron in 2017, this signature mimics what’s observed in the neurons of a newborn as compared to those of a fetus.

More recently, Dr. Pasca and his colleagues have been working to create different brain regions and have them assemble into what they are calling brain assembloids. They have generated iPSC lines from individuals with a variety of psychiatric disorders. Notably, two postdoctoral scholars, Nina Huber, PhD, and Fikri Birey, PhD, were each awarded $50,000 for their efforts in the Pasca lab.

“We’re now looking at things that in principle have the capacity to make little circuits and interact with each other,” Dr. Dolmetsch says. This is important because “a lot of diseases of the brain are not diseases of just one cell; they're diseases of multiple cell types.”

Dr. Dolmetsch describes his former mentee as the “guru of 3D cell models.” It’s an appropriate title. Dr. Pasca, just 36, is quickly becoming a leader in this new field of molecular psychiatry. In March, he was awarded the prestigious Vilcek Prizes for Creative Promise in Biomedical Science. Established in 2009, these awards of $50,000 honor early or mid-career foreign-born scientists who have already done exceptional work. (Watch a video on Dr. Pasca made by the Vilcek Foundation.) In May 2018, he was recognized by The New York Times as a Visionary who is using medicine and science to improve the quality of life. He also recently spoke about his work at CHRI’s Childx 2018.

Meanwhile, Dr. Dolmetsch has been the Global Head of Neuroscience at Novartis since 2013, when the company offered him an opportunity to parlay his research into finding treatments for intractable neuropsychiatric disorders in both children and adults. He made the leap to industry after nearly 10 years at Stanford, recognizing it would be hard to pursue this line of work in academia. Before Dr. Dolmetsch came onboard Novartis had not had a neuroscience department. Today, Dr. Dolmetsch directs projects from their earliest phases to proof of concept.

The two researchers are still working together—now on a project exploring 22q11.2 deletion syndrome, the most common genetic cause of schizophrenia and autism. They still occasionally have late night conversations, problem solving and brainstorming despite living on different coasts.  

All told, CHRI invested $371,052 toward advancing this fledgling field through the work of Drs. Dolmetsch and Pasca. It facilitated the formation of a partnership between the two, and afforded them the time and support needed to wrestle with the big question of how to obtain brain cells for research on the mechanisms of mental disorders.

While acknowledging the challenge of facing so many new unknowns, Dr. Pasca says he’s looking forward to the future of this field.  He believes “this is just the start of what one could call a molecular revolution in psychiatry.”

Laura Hedli is a writer for the Division of Neonatal and Developmental Medicine in the Department of Pediatrics and contributes stories to the Stanford Child Health Research Institute.