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I want to know if qualitative experiments have been done growing chimpanzee neurons and human neurons in vitro and have any differences emerged, such as the amount of connections per neuron or anything else.
I have came across several studies indicating large similarities between chimpanzee and humans. A study by Bianchi et al., 2013 used immunohistochemistry, electron microscopy, and Golgi staining to characterise synaptic density and dendritic morphology of pyramidal neurons in primary somatosensory (area 3b), primary motor (area 4), prestriate visual (area 18), and prefrontal (area 10) cortices of developing chimpanzees (Pan troglodytes). They found that synaptogenesis occurs synchronously across cortical areas, with a peak of synapse density during the juvenile period (3-5 y). Moreover, similar to findings in humans, dendrites of prefrontal pyramidal neurons developed later than sensorimotor areas. These results suggest that evolutionary changes to neocortical development promoting greater neuronal plasticity early in postnatal life preceded the divergence of the human and chimpanzee lineages. This study however does note that
Despite sharing these neurodevelopmental similarities, it is important to note that cognitive ontogeny in chimpanzees differs from humans in several respects. Behavioral studies suggest that the different social and environmental contexts in which humans and chimpanzee develop may have also been important in the evolution of human-specific socio-cognitive abilities. For example, whereas young chimpanzees do not fully wean until 4-5 y of age and remain closely attached to their mothers during the early years of development, human infants often interact with multiple caregivers and engage in joint attention.
In another study by Bianchi et al., also published in 2013 regional variation in the morphology of pyramidal neurons in the cerebral cortex of great apes, humans' closest living relatives were investigated. The study used the rapid Golgi stain to quantify the dendritic structure of layer III pyramidal neurons in 4 areas of the chimpanzee cerebral cortex: Primary somatosensory (area 3b), primary motor (area 4), prestriate visual (area 18), and prefrontal (area 10) cortex. The study showed that
Consistent with previous studies in humans and macaque monkeys, pyramidal neurons in the prefrontal cortex of chimpanzees exhibit greater dendritic complexity than those in other cortical regions, suggesting that prefrontal cortical evolution in primates is characterized by increased potential for integrative connectivity.
Compared with chimpanzees, the pyramidal neurons of humans had significantly longer and more branched dendritic arbors in all cortical regions.
These studies show that despite significant morphological and developmental similarities between human and chimpanzee neurons, there are still notable observable differences.
Scientists Grew Human Cells in Monkey Embryos, and Yes, It’s an Ethical Minefield
Few things in science freak people out more than human-animal hybrids. Named chimeras, after the mythical Greek creature that’s an amalgam of different beasts, these part-human, part-animal embryos have come onto the scene to transform our understanding of what makes us “human.”
If theoretically grown to term, chimeras would be an endless resource for replacement human organs. They’re a window into the very early stages of human development, allowing scientists to probe the mystery of the first dozen days after sperm-meets-egg. They could help map out how our brains build their early architecture, potentially solving the age-old question of why our neural networks are so powerful—and how their wiring could go wrong.
The trouble with all of this? The embryos are part human. The idea of human hearts or livers growing inside an animal may be icky, but tolerable, to some. Human neurons crafting a brain inside a hybrid embryo—potentially leading to consciousness—is a horror scenario. For years, scientists have flirted with ethical boundaries by mixing human cells with those of rats and pigs, which are relatively far from us in evolutionary terms, to reduce the chance of a mentally “humanized” chimera.
This week, scientists crossed a line.
In a study led by Dr. Juan Carlos Izpisua Belmonte, a prominent stem cell biologist at the Salk Institute for Biological Studies, the team reported the first vetted case of a human-monkey hybrid embryo.
Reflexive shudder aside, the study is a technological tour-de-force. The scientists were able to watch the hybrid embryo develop for 20 days outside the womb, far longer than any previous attempts. Putting the timeline into context, it’s about 20 percent of a monkey’s gestation period.
Although only 3 out of over 100 attempts survived past that point, the viable embryos contained a shockingly high amount of human cells—about one-third of the entire cell population. If able to further develop, those human contributions could, in theory, substantially form the biological architecture of the body, and perhaps the mind, of a human-monkey fetus.
I can’t stress this enough: the technology isn’t there yet to bring Planet of the Apes to life. Strict regulations also prohibit growing chimera embryos past the first few weeks. It’s telling that Izpisua Belmonte collaborated with Chinese labs, which have far fewer ethical regulations than the US.
But the line’s been crossed, and there’s no going back. Here’s what they did, why they did it, and reasons to justify—or limit—similar tests going forward.
What They Did
The way the team made the human-monkey embryo is similar to previous attempts at half-human chimeras.
Here’s how it goes. They used de-programmed, or “reverted,” human stem cells, called induced pluripotent stem cells (iPSCs). These cells often start from skin cells, and are chemically treated to revert to the stem cell stage, gaining back the superpower to grow into almost any type of cell: heart, lung, brain…you get the idea. The next step is preparing the monkey component, a fertilized and healthy monkey egg that develops for six days in a Petri dish. By this point, the embryo is ready for implantation into the uterus, which kicks off the whole development process.
This is where the chimera jab comes in. Using a tiny needle, the team injected each embryo with 25 human cells, and babied them for another day. “Until recently the experiment would have ended there,” wrote Drs. Hank Greely and Nita Farahany, two prominent bioethicists who wrote an accompanying expert take, but were not involved in the study.
But the team took it way further. Using a biological trick, the embryos attached to the Petri dish as they would to a womb. The human cells survived after the artificial “implantation,” and—surprisingly—tended to physically group together, away from monkey cells.
The weird segregation led the team to further explore why human cells don’t play nice with those of another species. Using a big data approach, the team scouted how genes in human cells talked to their monkey hosts. What’s surprising, the team said, is that adding human cells into the monkey embryos fundamentally changed both. Rather than each behaving as they would have in their normal environment, the two species of cells influenced each other, even when physically separated. The human cells, for example, tweaked the biochemical messengers that monkey cells—and the “goop” surrounding those cells—use to talk to one another.
In other words, in contrast to oil and water, human and monkey cells seemed to communicate and change the other’s biology without needing too much outside whisking. Human iPSCs began to behave more like monkey cells, whereas monkey embryos became slightly more human.
Ok, But Why?
The main reasons the team went for a monkey hybrid, rather than the “safer” pig or rat alternative, was because of our similarities to monkeys. As the authors argue, being genetically “closer” in evolutionary terms makes it easier to form chimeras. In turn, the resulting embryos also make it possible to study early human development and build human tissues and organs for replacement.
“Historically, the generation of human-animal chimeras has suffered from low efficiency,” said Izpisua Belmonte. “Generation of a chimera between human and non-human primate, a species more closely related to humans along the evolutionary timeline than all previously used species, will allow us to gain better insight into whether there are evolutionarily imposed barriers to chimera generation and if there are any means by which we can overcome them.”
A Controversial Future
That argument isn’t convincing to some.
In terms of organ replacement, monkeys are very expensive (and cognitively advanced) donors compared to pigs, the latter of which have been the primary research host for growing human organs. While difficult to genetically engineer to fit human needs, pigs are more socially acceptable as organ “donors”—many of us don’t bat an eye at eating ham or bacon—whereas the concept of extracting humanoid tissue from monkeys is extremely uncomfortable.
A human-monkey hybrid could be especially helpful for studying neurodevelopment, but that directly butts heads with the “human cells in animal brains” problem. Even when such an embryo is not brought to term, it’s hard to imagine anyone who’s ready to study the brain of a potentially viable animal fetus with human cells wired into its neural networks.
There’s also the “sledgehammer” aspect of the study that makes scientists cringe. “Direct transplantation of cells into particular regions, or organs [of an animal], allows researchers to predict where and how the cells might integrate,” said Greely and Farahany. This means they might be able to predict if the injected human cells end up in a “boring” area, like the gallbladder, or a more “sensitive” area, like the brain. But with the current technique, we’re unsure where the human cells could eventually migrate to and grow.
Yet despite the ick factor, human-monkey embryos circumvent the ethical quandaries around using aborted tissue for research. These hybrid embryos may present the closest models to early human development that we can get without dipping into the abortion debate.
In their commentary, Greely and Farahany laid out four main aspects to consider before moving ahead with the controversial field. First and foremost is animal welfare, which is “especially true for non-human primates,” as they’re mentally close to us. There’s also the need for consent from human donors, which form the basis of the injected iPSCs, as some may be uncomfortable with the endeavor itself. Like organ donors, people need to be fully informed.
Third and fourth, public discourse is absolutely needed, as people may strongly disapprove of the idea of mixing human tissue or organs with animals. For now, the human-monkey embryos have a short life. But as technology gets better, and based on previous similar experiments with other chimeras, the next step in this venture is to transplant the embryo into a living animal host’s uterus, which could nurture it to grow further.
For now, that’s a red line for human-monkey embryos, and the technology isn’t there yet. But if the surprise of CRISPR babies has taught us anything, it’s that as a society we need to discourage, yet prepare for, a lone wolf who’s willing to step over the line—that is, bringing a part-human, part-animal embryo to term.
“We must begin to think about that possibility,” said Greely and Farahany. With the study, we know that “those future experiments are now at least plausible.”
Image Credit: A human-monkey chimera embryo, photo by Weizhi Ji, Kunming University of Science and Technology
Brain waves detected in mini-brains grown in a dish
Pea-size brain organoids at 10 months old. Credit: Muotri Lab/UCTV
Scientists have created miniature brains from stem cells that developed functional neural networks. Despite being a million times smaller than human brains, these lab-grown brains are the first observed to produce brain waves that resemble those of preterm babies. The study, published August 29 in the journal Cell Stem Cell, could help scientists better understand human brain development.
"The level of neural activity we are seeing is unprecedented in vitro," says Alysson Muotri, a biologist at the University of California, San Diego. "We are one step closer to have a model that can actually generate these early stages of a sophisticated neural network."
The pea-sized brains, called cerebral organoids, are derived from human pluripotent stem cells. By putting them in culture that mimics the environment of brain development, the stem cells differentiate into different types of brain cells and self-organize into a 3-D structure resembling the developing human brain.
Scientists have successfully grown organoids with cellular structures similar to those of human brains. However, none of the previous models developed human-like functional neural networks. Networks appear when neurons are mature and become interconnected, and they are essential for most brain activities.
"You can use brain organoids for several things, including understand normal human neurodevelopment, disease modeling, brain evolution, drug screening, and even to inform artificial intelligence," Muotri says.A cross-section of a brain organoid, showing the initial formation of a cortical plate. Each color marks a different type of brain cell. Credit: Muotri Lab/UCTV
Muotri and colleagues designed a better procedure to grow stem cells, including optimizing the culture medium formula. These adjustments allowed their organoids to become more mature than previous models. The team grew hundreds of organoids for 10 months and used multi-electrode arrays to monitor their neural activities.
The team began to detect bursts of brain waves from organoids at about two months. The signals were sparse and had the same frequency, a pattern seen in very immature human brains. As the organoids continued to grow, they produced brain waves at different frequencies, and the signals appeared more regularly. This suggests the organoids have further developed their neural networks.
"This is a result of having more functional synapses, and you are forming more connections between the neurons," Muotri says. The interactions between neurons contribute to signals at various frequencies, he says.
To compare the brain wave patterns of organoids with those of human brains early in development, the team trained a machine learning algorithm with brain waves recorded from 39 premature babies between six and nine-and-a-half months old. The algorithm was able to predict how many weeks the organoids have developed in culture, which suggests these organoids and human brain share a similar growth trajectory.This activity map generated by multi-electrode arrays reveals how active the brain organoid is--red means very active and black means silent. Credit: Muotri Lab/UCTV
However, it's not likely these organoids have mental activities, such as consciousness, Muotri says. "The organoid is still a very rudimentary model—we don't have other brain parts and structures. So these brain waves might not have anything to do with activities in real brains."
"It might be that in the future, we will get something that is really close to the signals in the human brains that control behaviors, thoughts, or memory," Muotri says. "But I don't think we have any evidence right now to say we have any of those."
Looking forward, the team aims to further improve the organoids and use them to understand diseases associated with neural network malfunctioning, such as autism, epilepsy, and schizophrenia.
"As a scientist, I want to get closer and closer to the human brain," Muotri says. "I want to do that because I see the good in it. I can help people with neurological conditions by giving them better treatments and better quality of life. But it's up to us to decide where the limit is. It might be that the technology is not ready yet, or we don't know how to control the technology. This is the same kind of discussion around CRISPR in babies, and that's why we have ethics committees to represent all parts of the society."
Diseases In A Dish: Modeling Mental Disorders
Using skin cells from patients with mental disorders, scientists are creating brain cells that are now providing extraordinary insights into afflictions like schizophrenia and Parkinson’s disease.
The Researcher s
For many poorly understood mental disorders, such as schizophrenia or autism, scientists often wish they could turn back the clock to uncover what has gone wrong in the brains of these patients, and how to right it before much brain damage ensues. But now, thanks to recent developments in the lab, that wish is coming true.
Left to right: Fred Gage, a professor of genetics at the Salk Institute for Biological Studies and member of the executive committee of the Kavli Institute for Brain and Mind (KIBM) at the University of California, San Diego (UCSD), and Anirvan Ghosh, neurobiologist at UCSD and also an executive committee member of KIBM.
Researchers are using genetic engineering and growth factors to reprogram the skin cells of patients with schizophrenia, autism, and other neurological disorders and grow them into brain cells in the laboratory. There, under their careful watch, investigators can detect inherent defects in how neurons develop or function, or see what environmental toxins or other factors prod them to misbehave in the petri dish. With these “diseases in a dish” they can also test the effectiveness of drugs that can right missteps in development, or counter the harm of environmental insults.
“It’s quite amazing that we can recapitulate a psychiatric disease in a petri dish,” says neuroscientist Fred (Rusty) Gage, a professor of genetics at the Salk Institute for Biological Studies and member of the executive committee of the Kavli Institute for Brain and Mind (KIBM) at the University of California, San Diego. “This allows us to identify subtle changes in the functioning of neuronal circuits that we never had access to before.”
Below is an edited transcript of a conversation with Gage and Anirvan Ghosh, a neurobiologist at the University of California, San Diego and also an executive committee member of KIBM. Both researchers are on the cutting edge of disease-in-a-dish modeling of neurological disorders. Gage and Ghosh discuss how human skin cells induced to return to an immature state (“induced pluripotent stem cells” or IPS cells) are revolutionizing our understanding and treatment of mental and neurodegenerative disorders, such as Parkinson’s disease, as well as leading to new models of drug development for all diseases.
THE KAVLI FOUNDATION (TKF):Dr. Gage, in your model you found that neurons look pretty similar between schizophrenic patients and normal controls, and it is just the connections between them (synapses) that are different, right?
FRED "RUSTY" GAGE: Yes. That doesn’t mean on closer inspection, and with better tools, more profound or subtle changes won’t be found. I wouldn’t be surprised if some more specific defects are revealed by a more sophisticated tool.
GAGE: We have to admit to ourselves this is a model of what might be going on in the brain and not think of it as a one-to-one relationship. It’s too soon to ask if this is how it happens in a patient instead we are asking how do these drugs affect neurons.
TKF:So what are some of the surprises this modeling has revealed so far?
GAGE: One surprise is that neurons appear to undergo structural changes when they are given neuropsychiatric drugs. This is unexpected, as since the 1970’s companies have developed neuropsychiatric drugs on the premise that you modulate mood by regulating the amount of chemical signals available in the brain. These chemical signals are called neurotransmitters, and consequently the drugs have focused on modulating neurotransmitters such as dopamine and serotonin.
But one of the take home messages I got from my study is it’s not just the moment-to-moment regulation of dopamine that may be affecting the symptoms of schizophrenia, but the structural organization of how these synapses interact with each other. In other words, changing the regulation of dopamine or some other compound appears to have the additional effect of causing structural modifications that also affect how neurons interact.
TKF: This might explain why many of these drugs require a long period of time before a patient experiences a benefit.
GAGE: Exactly. If depression is merely due to a modulation of transmitter content and its receptor affinity, why wouldn’t these anti-depressants have their effects immediately? Normally it takes weeks. One of the emerging ideas is that part of the reason it takes longer is these drugs have other effects than what we’ve anticipated. Our findings support that possibility. Looking ahead, the next generation of drugs may not target dopamine or serotonin concentrations but instead the structure and function of synapses.
TKF: Dr. Ghosh, what other surprises has this modeling revealed?
ANIRVAN GHOSH: I think it’s remarkable what Rusty uncovered about schizophrenia. This is a pretty broad disorder and one we've suspected may have many different genetic causes. Yet they found there were shared cellular traits (phenotype) between the patients. This is a very exciting result because it raises the possibility of being able to find the phenotype—physical hallmarks--that might be shared for most, if not all, individuals with the disease.
TKF: What technological advances are needed to explore this further?
GAGE: One limitation is we haven’t differentiated the cells into specific cell types—neuronal subtypes. Right now we’re just laying these neurons down and allowing them to form connections as they might. Looking ahead, it’s going to be important for us to differentiate the cells. For example, to differentiate and model the cortical neurons, which are responsible for thinking tasks, or the hippocampal neurons, which are responsible for memory tasks. I can one day see us using microfluidic chambers to achieve this. They will allow us to compartmentalize microscopically specific subtypes of neurons in certain locations, and then regulate how they connect to each other. That way you can simulate in a more accurate manner how these subtypes connect with each other in the brain. The future of this is really exciting because the dish is going to get much more complicated.
TKF: So you’ll be able to combine the "disease in a dish" with a "lab on a chip"?
GAGE: Yes, the bioengineering part of this is becoming very exciting and interesting, and a lot of us will be relying heavily on that. It will enable three-dimensional cultures so you can have dopamine neurons projecting through a gradient into the types of neurons affected by Parkinson’s disease, for example. This way you can set up a whole neuronal network or circuitry that hopefully will be akin to what you see in the brain. We’re also trying to figure out what role inflammation plays in Parkinson’s disease. So for our model of this disease, which we are currently developing, we have to generate a variety of other brain cell types besides neurons that are thought to foster immune responses in the brain.
TKF: And you can compare mental disorders literally side-by-side or rather, dish-by-dish.
GAGE: That’s the idea. As we accumulate models for these diseases — bipolar disease, schizophrenia, depression, autism — we are going to be able to explore if there are really differences between them that exist on a cellular or gene expression level.
Human neurons differentiated from skin-derived stem cells. Comparison of neurons from unaffected individuals and patients could provide insight into the underlying causes of neurological and psychiatric disorders. (Credit Ji-Eun Kim and Anirvan Ghosh, UCSD)
GHOSH: I’m also really excited about using this platform to stratify patient groups and develop therapies that are more appropriate for them. For example, there are many causes for autism, so the same drug may not work for all patients with this disorder. But we could see how the cultured neurons of these patients respond to a particular drug or stimulus. Based on that, we could then classify them into specific groups and thereby provide a treatment that would be more effective for them. One can imagine down the road in a couple of years, if you have a child with autism, you could use this kind of platform to determine what kind of subtype he falls into, which would influence what kind of treatment he’ll get.
GAGE: I agree. There is going to be an exciting interface between basic scientists and clinicians. In schizophrenia, for example, it’s not unusual for a physician to test three or four different psychoactive drugs before they find the one a patient can minimally respond to. But suppose we could stratify patients to select the most likely appropriate drug based on their own brain cells and how they function? Furthermore, this will help us understand mechanistically how and why some patients may respond to the same drugs and others do not.
TKF: It seems the use of more selective and targeted drugs could have a revolutionary effect on the drug industry. What sense do you have of the support by drug manufacturers for this type of modeling?
GHOSH: There’s a lot of interest in using this as a platform for drug screens and drug discovery, and not necessarily waiting for five years to see how it develops. This is pretty surprising and quite bold, because many of the papers that report differences are incredibly recent and haven’t been reported in multiple labs. Normally industry tends to be pretty conservative about getting into these sorts of things. In the past, when we’ve talked to various pharmaceutical companies about one program or another in our lab, they often want to wait until it’s been developed into a preliminary screen, and would rather work with biotech companies for collaborations—they’ve often been the intermediate window before a pharmaceutical company would step in. But in this case, Roche and I have been active in directly interacting with various academic groups.
GAGE: I see it too. The pharmaceutical industry is really getting interested and supporting more interactions between basic laboratories and their own work. There is a tighter link between the basic science and the clinical science labs. We talk to these guys all the time now about the patients, which is really amazing. By working with clinicians caring for patients, we basic scientists are gaining a lot of inside information. The clinicians are also benefiting and now more and more clinicians are calling up and saying, ‘We’ve got this really interesting group of patients, and we’d love to see whether or not you could do something with them.’ We’re beginning to formulate specific hypotheses about what might be there, given what we know now. It’s striking that we’re already on the bridge between the lab and the clinic. It’s also exciting to see the young people coming into the field. These people, who are trained in cell biology, molecular biology, basic physiology or in other specialties, are now able to contribute to specific disease-related studies in a really viable way.
TKF: How is this new dynamic being fostered?
GHOSH: The interactions between basic science labs, industry and patient foundations are changing and proving very productive, and from the beginning, the California Institute for Regenerative Medicine has had a huge influence. It’s getting many people into this area and exploring things that otherwise they wouldn’t have, our lab included. The foundations – which are often associated with specific diseases –have also played a very positive role in bringing together all these different groups and getting them to work effectively together. In fact, the patient foundations are incredibly important because they are the most effective in making the case for supporting this research at places like NIH and Congress.
Thanks to all of this, today there is more talk about how one goes from basic science observations to potential therapies. It’s an unusual kind of alliance that has evolved out of this human stem cell work and does not exist so much in other areas of biology. It’s a new and really exciting model to get scientists to work with groups that are involved in drug development.
TKF: What sorts of more basic insights is this modeling providing?
GHOSH: We’re learning a lot about development of normal human neurons, which would have been impossible previously, so there’s a deep knowledge base in terms of understanding how cells mature and differentiate, how synapses behave, and perhaps circuits involving neurons behave. This is going to lead to a deep baseline understanding of various disorders. There also might be certain disorders you can treat by transplanting a small number of differentiated cells, so this knowledge would be useful for that. Understanding the pathways that allow the cells to divide or proliferate could also be used to manipulate cells in a particular aspect of the nervous system. There are populations of cells that divide in the mature brain, and the knowledge we gain from the in vitro studies could help influence how one might be able to manipulate and use that newly generated population of cells in the brain in more therapeutic ways. Those haven’t been explored much, but could be useful in the future.
GAGE: I think it will also give us insights into issues related to evolution. We basically have an entire zoo of neurons in a dish now, so with these models we can begin to look at underlying mechanisms that may be different between neurons of different species, and explore what is the basis of these differences between species. We can even look at our closer relatives and get to the essence of what makes us human.
Human Brains Growing in Lab Aren’t Thinking … Yet
Researchers in England are growing small human brains in their laboratories. Even more lab-grown-mind-boggling, they grew the brains out of human skin cells. Are these tiny brains thinking that this is pretty amazing? Not yet, but some scientists are concerned that the day is coming.
While mini-brains have been grown from stem cells for testing drugs or studying the effects of the Zika virus, this is the first time brains have been grown from non-stem cells for the purpose of studying how and why human brains are superior to the brains of other primates even though our DNA is only 1.,2 percent different from the DNA of chimpanzees. That’s according to a recent BBC Future interview with Madeline Lancaster, the research leader at the Medical Research Council (MRC) Laboratory of Molecular Medicine in Cambridge, England.
In an effort to better understand human brain development, we have developed a new model system, called cerebral organoids. Cerebral organoids, or mini-brains for short, are 3D tissues generated from human pluripotent stem cells that allow modelling of human brain development in vitro.
The cerebral organoids are really mini – only 4 millimeters across – and their development is fascinating … and frightening. Ordinary human skin cells are immersed in a kind of protein shake that causes them to grow as if they were embryonic again – only skin cells have this ability. A ball of these newly-created embryonic stem cells are placed in a Petri dish where they each begin to diversify into various body part cells. At that point, this bigger ball of cells is placed in another Petri dish containing almost no food, effectively starving the cells to death – all except the brain cells, which Lancaster says “… are really robust – I don’t think anyone knows why.”
So now they have a Petri dish of hardy survivalist brain cells. These are rewarded with a new Petri dish (this lab must make its Petri dish salesperson very rich) filled with a special jelly that acts like an embryonic skull – feeding, nurturing and molding the cells for three months until they become a tiny embryonic brain, which Lancaster says is full of firing neurons.
It’s not very special but it does tell us that we are making functional neurons and that they are acting like neurons.
Cross-section of mini-brain
Does that mean the tiny brain is thinking? Lancaster says no, and she promises that her lab will only use them as is to study neurological conditions such as autism and schizophrenia.
This is kind of a good thing, I think. I’d have some issues if I thought there was proper network formation there.
However, she admits that the next step is to grow chimpanzee brains from skin cells, which would have less ethical restrictions. Dr. Martin Coath from the Cognition Institute at the University of Plymouth warns that other scientists may not be as principled as Lancaster.
Something we have grown in the lab, but on a much simpler level than a human brain, might be hooked up to electronic eyes, ears, and hands and be taught to do something – maybe something that is as sophisticated as many simple living creatures. That doesn’t seem so far off to me.
A human brain that was ‘fully working’ would be conscious, have hopes, dreams, feel pain, and would ask questions about what we were doing to it.
What answer would they give? What answer SHOULD they give? Would it be too late. Is it already too late?
Opening skinner's box - chapter 9 (from on-line)
→→ studied nerve cells in the hippocampus, which is hard to work with so he decided to work on the giant marine snail aplysia -- slug as they only have 20,000 neurons, many of which are visible to the eye and nervous system is the same as humans
→ Kandel trained slug -- touched their goopy bodies with an electric probe and the sea slug's gill withdrew
→→ soon discovered that this reflex could be modified but 3 different forms of learning
→→ its neurons changed -- the synapses grew stronger by passing electrochemical signals that reinforced the relationship
→ then showed that by blocking cAMP-response element binding protein (CREB)(a tiny molecule deep in nerve cell 1), he could disrupt the convo
→→ closing in on a new class of drugs that promises to revise our notions of age, time
→→ goal - find a chemical compound that will help the disembodied neurons in the petri dish and the embodied neurons in the mind form stronger, longer lasting connections
→ what happens if the drug somehow loosens the lids of our archives
→→ might trap us in such a detailed past that we cannot focus on where we are
Harvard neuroscientist Venkatesh N. Murthy has a sunny second-floor office on Divinity Avenue, where he is a professor in Harvard’s Department of Molecular and Cellular Biology. In one corner is a set of weights and a soccer ball — both untouched in over a year, he said, because of an intensely busy schedule.
There’s a stack of jazz CDs — the 43-year-old Murthy is a fan, and admits to playing jazz guitar badly — and there’s a white board covered with arcane scrawls about his specialty: how brain cells connect with one another.
It’s a specialty involving vast numbers. There are an estimated 100 billion neurons in the average 3-pound human brain. Connecting them are as many as 10 trillion synapses, the circuitlike chemical pathways that link neurons to one another. “The power of higher brain areas,” said Murthy, “is in numbers.”
The numbers give neurons and the brain immense computational power, he said. In turn, the brain’s plasticity (functional flexibility) comes in part from synapses that can be big, small, weak, strong — a range of variations in the trillions.
Finding out how synapses grow, fire, modify, and break is important work. The synaptic impulses that link neurons are vital they transform brain activity into motion by delivering messages from the brain and spinal cord to muscles and organs.
Yet the actual mechanisms of synaptic connectivity, at the cellular level, are “largely mysterious,” said Murthy — “Venki” to his friends. He has been at Harvard since 1999, arriving from postdoctoral work at the prestigious Salk Institute for Biological Studies. Only in the past decade, said Murthy, have scientists “begun to draw a reasonable cartoon” of how synapses work — how they grow, load up with the right chemicals, pass on information, communicate with one another, and get recycled.
Better understanding of how synapses work could one day have profound implications for the treatment of diseases affected by neural impulses. Included are Parkinson’s, autism, depression, and schizophrenia. (Mutations in certain genes tied to synaptic function have been linked to schizophrenia, whose genetic origins are an interest of Murthy’s.)
He and his research team are using the brains of mice to model how synapses work. Specifically, they are taking real-time pictures of the way synapses light up in a region of the brain called the olfactory bulb, where odor information is processed.
Eventually, they’ll explore the way synapses are altered by experience in a deeper and more complex region of the brain called the cortex.
In the meantime, the olfactory bulb as a model offers many advantages. It’s close to the surface of the skull, eliminating the need for imprecise and invasive probes. And it’s a sensory system — meaning that it has a known set of responses to known stimuli. A smell produces a predictable reaction within a discrete location of the brain.
But observing and measuring synaptic connectivity even in the accessible and transparent olfactory system is a technical challenge. For one, neurons are tiny, measured in millionths of a meter — each about 10 microns across. For another, their related synapses fire in transient bursts, covering just fractions of a second.
So Murthy and his researchers have built recording and measurement gear from scratch. In several labs there are combinations of powerful multiphoton microscopes optical microscopy arrays that record synaptic firing and download it into computers as images and machines that deliver sequential odor stimuli in precise doses to the noses of rodents.
In one lab, one such olfactometer sprouts 100 tubes, each capable of measuring out precise puffs of smell from synthetic chemicals. (Some of the odors are familiar, like camphor and spearmint. Most are synthetic chemicals known to light up the olfactory center.) The odor signals go to a mouse, whose cascade of changing brain reactions are captured on a computer screen.
In his office, Murthy presses a few buttons on his laptop. A video appears, showing a rodent skittering around a tiny box blanketed with an odorant-soaked paper towel. Attached to the mouse’s head is an array of hairlike flexible wires a meter long. They transmit, in real time, images of the mouse’s reaction to odor stimulation.
The skittering rodent is breaking scientific ground. So will the mouse soon to run in place atop a rotating plastic ball, in a laser-based measuring device being constructed in another Murthy lab. Ordinarily, observing synaptic activity is done in anesthetized animals, not ones that are alert, awake, and reacting to their surroundings.
In his first years at Harvard, Murthy continued his Salk Institute experiments, studying synaptic activity in vitro by watching how nerve cells from the hippocampus, isolated in a Petri dish, react to stimuli. That’s good for understanding the “detailed mechanisms” of synapse biochemistry, he said. But there’s no substitute for in vivo research — looking at “synapses during the actual behavior in the actual animal,” said Murthy. “We want to understand [synaptic connectivity] in the context of the real thing.”
Natural experiments like this, in monkeys, occupied Murthy when he was a doctoral student in physiology and biophysics at the University of Washington, Seattle.
Murthy’s doctoral thesis on nerve impulses in the cortex was a hit in the scientific press. He and co-researchers observed that a pattern of coherent (or synchronous) brain activity occurred more frequently during novel untrained behavior. The hypothesis: This mode of activity may coordinate multiple regions of the brain during complex behaviors that require attention. His dissertation, said Murthy, “is still my most-cited paper, and also my oldest in neuroscience.”
The India-born researcher admits to having taken — in academic terms — a rather eccentric path to get to where he is. His father was an engineer from a family of engineers, and Murthy himself — a gifted student — landed a coveted spot as an undergraduate in mechanical engineering at the Indian Institute of Technology in Chennai (Madras) in his native South India.
India’s seven Indian Institutes of Technology, including the one in Chennai, consider around 100,000 applications a year 4,000 of these applicants are admitted.
“Once you get in, it’s hard not go there,” he said of the school — where his graduating class (1986) produced two other current Harvard professors: Ananth Raman (Harvard Business School) and L. Mahadevan (Harvard University School of Engineering and Applied Sciences).
He took a master’s degree in bioengineering in Washington, “but I was still writing equations and solving them for someone else,” said Murthy. “I still didn’t understand what science meant — trying to understand how something works, asking the questions, then finding the solutions.”
He transferred to the doctoral program in biophysics and physiology. At age 25, said Murthy, he was finally ready to explore science fully, including his first course work in biology.
By the time he left Seattle, he had settled on his life’s work, a cellular-level analysis of neurons. Salk “was the best I could have done,” said Murthy — a multidisciplinary immersion in how synapses work. Synaptic transmission “is an elementary event, (common) to all animals,” he said, calling it a still-mysterious arena of “exquisite regulation.”
At home in Newton, Mass., there’s exquisite regulation of a sort too. The onetime graduate school soccer player and touring bicyclist is getting a workout raising his two daughters (Sophie, 4, and Sonia, 1) with his biologist wife Meredith, a biotech clinical trials specialist. Murthy is between two busy worlds — long hours of research and deep hours of child care.
Luckily, there is coffee. An espresso machine ticks away in the anteroom to his office, where graduate students catch a bite and read. “I admit,” said Murthy, fussing with a china mug, “to being slightly addicted.”
Disorder in a dish: PhD alum Sundari Chetty uses human cells to study autism and schizophrenia
To understand the mechanisms underlying disorders such as autism and schizophrenia, Sundari Chetty first takes blood or skin cells from patients and induces them to become stem cells. Then she coaxes these so-called induced pluripotent stem cells (iPSCs) to produce different types of mature brain cells, allowing her to model the disorders in a petri dish where she can even test potential treatments.
Chetty is an assistant professor of psychiatry and behavioral sciences at Stanford University, and an alum of the Berkeley Neuroscience PhD Program. She was an undergraduate molecular and cell biology major at UC Berkeley, where she discovered her passion for therapeutically-relevant neuroscience research as a student in Robert Knight’s lab . Knight is a neurologist and a member of the Helen Wills Neuroscience Institute.
Chetty’s exposure to neuroscience at Berkeley inspired her to join the Neuroscience PhD Program. She did her PhD in Daniela Kaufer’s lab , where she studied how stress alters the fate of stem cells in the hippocampus of the adult rat brain and how that may affect learning and memory. She then went on to do a postdoctoral fellowship in Douglas Melton’s lab at Harvard, where she studied the mechanisms regulating differentiation and cell fate choice of human embryonic stem cells and iPSCs. After her postdoc, she started her own lab at Stanford in 2016.
Read our Q&A with Chetty to learn how she uses human cells to search for new treatments for neurological disorders why she liked the atmosphere of neuroscience at Berkeley and how being a parent meshes well with being a developmental neuroscientist. This Q&A has been edited for length and clarity.
Rachel Henderson: How did you become interested in neuroscience?
Chetty and her daughter on Halloween.
Sundari Chetty: I grew up in a family of doctors. My father is a physician as well as my two older sisters and younger brother. So I was always very interested in medicine and the science behind it. I became interested in research when I was doing my undergraduate degree at UC Berkeley. I was in the molecular and cell biology [MCB] department, emphasizing in neurobiology. I was fortunate to get exposure to neuroscience research in Bob Knight’s lab , which really sparked my interest in doing research that has therapeutic relevance. I really wanted to do something that was translational — to bridge my interest in science and medicine together so that I could possibly find new therapies for some of the neurodegenerative disorders or neurobiological issues affecting mental health.
RH: Why did you choose the Neuroscience PhD Program at Berkeley?
SC: I went to Berkeley for my undergraduate degree and especially in my last few years, I was exposed to a lot of the neuroscience research, both in MCB as well as through cognitive neuroscience by working in Bob Knight’s lab. That had really sparked my interest in wanting to understand how the brain functions and works, and how that can affect normal wellbeing as well as a lot of mental disorders or neurodegenerative disorders. Because I was at Berkeley and I knew the faculty and the atmosphere fairly well, I was very interested in doing a PhD at Berkeley.
I really liked the Neuroscience Program at Berkeley. It was so new at that time and seemed to have a lot of potential, including opportunities for collaboration with other labs across disciplines. I really liked that atmosphere of the science and it seemed like a very enriching program where you learn a lot, get exposed to new kinds of ways to tackle questions, and are able to integrate with different colleagues, even if you’re in one particular lab.
RH: What was your PhD thesis about?
SC: I did my PhD in Daniela Kaufer’s lab , where I investigated how stress affects the brain. In particular, we were looking at how stress affects hippocampal neurogenesis in adulthood. The hippocampus has a region where there are neural stem cells that have the ability to become neurons in adulthood and that has impacts on learning and memory. I was particularly interested in understanding the molecular and cellular mechanisms of how stress regulates that process. We used rat models as well as in vitro cell culture systems, where we isolated the neural stem cells out of the rat brains, cultured them, and exposed them to stress hormones, particularly corticosterone which is a rodent form of cortisol. What we found was that in the presence of high levels of corticosterone, the neural stem cells both in vitro as well as in vivo would turn into oligodendocytes [ Ed. note : oligodendocytes are glial cells that produce the myelin sheath surrounding the axons of neurons] over neurons. We suppressed generation of new neurons by supporting the oligodendrocyte path, which impacts myelination of the neurons, and these effects could be reversed if we blocked stress signaling mechanisms through the glucocorticoid receptor.
Chetty and her lab at Stanford.
The way we showed this in vivo was with a few different stress paradigms. We exposed the rats to restraint stress, and that elevates their stress hormones over the span of a week. Then we would isolate the brains and look at the neural stem cells embedded in the dentate gyrus of the hippocampus and examine the cell fate of those neural stem cells. In the in vivo model as well, we saw that the neural stem cells had suppressed generation of neurons and had more generation of oligodendocytes. Then we injected corticosterone directly into the animals and saw the same effects — there was an increase in oligodendrocyte generation over neurons. We began some preliminary studies to look at how this would impact function, or learning and memory. That was still at the early stages, but it seemed to have an effect on increasing long-term learning and memory retention, potentially having implications for disorders such as post-traumatic stress disorder.
RH: Because you had been an undergraduate at Berkeley, did you know Daniela Kaufer before you started in the Neuroscience PhD Program?
SC: No, I didn’t. Daniela actually joined UC Berkeley during my first year, so I only got to know of her work after she had arrived at Berkeley. Her lab was the fourth rotation that I did during my first year, but I really fell in love with it. I liked both the molecular and cellular mechanism aspect of it and how that translated to mental wellbeing or brain function. I really liked having the ability to study basic science and seeing how that might affect human behavior.
I actually did a couple of rotations in imaging and cognitive neuroscience, then moved to a more systems approach where I worked with primate models. Then I ended up in more of a molecular and cellular lab — Daniela’s lab. I did this whole spectrum mostly because I wasn’t sure which area I wanted to go into. But I was glad I got the exposure to all aspects of neuroscience.
RH: I noticed you have a publication with HWNI member William Jagust as well.
SC: That was from my rotation in Bill Jagust’s lab in the first year of my PhD. Each of my rotations was very fruitful. Even though it’s a short time span that you get in each of the rotations, I had the opportunity to really go deep into many of the projects, which was a very nice exposure to the wide array of techniques in the field.
RH: What was your experience in the Berkeley Neuroscience PhD Program like in general?
SC: I loved the Berkeley PhD program. I still have fond memories of it. The cohorts of students in each class were fantastic. We were a pretty small class of about 10 students and I’ve kept in touch with many of my classmates, who are either still doing academic research or have moved into industry. So it really formed nice connections and friendships that have lasted for a long time. It was a very motivated group of people to be around, who were enthusiastic and passionate about science. They were easy to talk to about ideas or anything that’s going on in your life.
I really liked the exposure to different fields, not just within neuroscience, but also other fields of molecular and cellular biology, mostly because HWNI is very tightly integrated with MCB. You get a chance to see the science behind many areas, and attend seminars and journal clubs.
We also had something called Neurofriends where the Neuroscience PhD Program allowed us to have these lovely lunches with our classmates. I think that really helped us form tight connections and let us hear about the work that our classmates were doing. Often they were not in the same area — some might be in imaging and cognitive neuroscience, while some were in a more systems area, and some were in more of a molecular and cellular biology area. It was really nice to have those opportunities to interact with your own classmates in a more informal setting.
RH: What did you do after you left Berkeley?
SC: I continued to be interested in stem cell research after my PhD in Daniela’s lab, and I really wanted to bring it more to therapeutic relevance, which is my long-term interest. At that time, human embryonic stem cell work was gaining a lot of momentum. I wanted to do research in induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) and hopefully bridge it back to neuroscience or neurodegenerative diseases. So I chose to go into a postdoc at Harvard in Doug Melton’s lab, where I studied the mechanisms that regulate cell fate of human ESCs and iPSCs.
Research from the Chetty lab.
One of the main challenges in the stem cell field is to efficiently and effectively guide different stem cell lines into a lineage of choice. Many barriers prevent ESCs and iPSCs from readily differentiating, greatly limiting their applications for disease modeling and therapy. During my postdoc, I focused on understanding the mechanisms that regulate ESC/iPSC differentiation and found that if you modulate cell proliferation and the cell cycle by enriching cells in the early G1 phase of the cell cycle, there was increased propensity for differentiation into any lineage of choice following directed differentiation. The reason this is important is because ESC/iPSCs have a truncated cell cycle with short gap phases that promote amplification over differentiation. However, by enriching cells in the G1 gap phase and allowing the cells to pause from cell division, ESCs/iPSCs were significantly more responsive to the differentiation signals in a dish. This work was along the lines of what I had done in Daniela’s lab looking at neural stem cells and how environmental triggers can alter cell fate. But here, I was focused more on unleashing the differentiation potential of embryonic and induced pluripotent stem cells into any lineage in the body for therapeutic applications.
Then I moved to Stanford to start my own lab. I’ve been using human iPSCs to study psychiatric disorders, particularly autism and schizophrenia. We’re using human iPSCs for disease modeling approaches, in the hopes that we can understand disorders like autism and schizophrenia using humanized models and also identify new therapeutics by screening for drugs in an in vitro system.
Some of our ongoing projects focus on studying autistic children, who have and do not have brain enlargement. As you know, autism spectrum disorder is quite heterogeneous. There are a lot of different subtypes of autism, typically affecting boys more than girls. About 20% of autistic children have an enlarged brain relative to their body height, and this has been associated with poor outcomes. They have more severe behavioral deficits, lower IQ, and they don’t seem to be as responsive to the standard therapeutic interventions. Even though one may try to treat these kids at higher intensity — because they are so severe on the spectrum — they still don’t seem to respond to the therapies that are currently available.
What we wanted to do was understand the basic mechanisms that may be contributing to this brain overgrowth in autistic children. We have an NIH-funded grant with UC Davis, where we are collecting blood samples or skin fibroblasts from kids with and without autism, who have normal brain volume or enlarged brains. My lab is currently generating iPSCs from those kids and differentiating the iPSCs into different brain cell types — microglia (the immune cells in the brain) as well as cortical neurons and cortical oligodendrocytes. This is because in the kids who have the brain enlargement, imaging studies have shown that there are increases in gray and white matter. We wanted to model these effects in vitro , so we’re generating both neurons for gray matter as well as oligodendrocytes that may model the changes in white matter.
Schematic showing the Chetty lab’s approach of using peripheral blood mononuclear cells (PBMCs) from blood samples of patients and control subjects to reprogram them into expandable populations of iPSCs that can subsequently be differentiated into neural progenitor cells (NPCs), oligodendrocyte progenitor cells (OPCs), and microglia following directed differentiation. The cellular and molecular mechanisms are correlated with brain imaging and behavioral testing data obtained on the same individuals to gain deeper understanding of the underlying mechanisms contributing to psychiatric and neurodevelopmental disorders and ultimately identify more targeted therapeutics.
We’re working with over fifty iPSC lines in the lab from different kids and patients with these types of neurodevelopmental and psychiatric disorders (with and without brain enlargement), differentiating the cells over a span of a month or two months to generate the different neural and glial cell types, and looking at what is contributing to the differences. We perform a lot of RNA sequencing, gene expression studies, protein analyses, and functional assays to gain understanding of the mechanistic insights.
One of our recent findings is that the brain overgrowth in some forms of autism (e.g. 16p11.2 deletion syndrome) is associated with overexpression of CD47, which is a ‘don’t eat me signal’ that’s upregulated in a lot of cancer cells, leading to suppressed engulfment by immune cells. We had hypothesized that perhaps the brain enlargement is due to improper elimination of cells early in development, and that may be contributing to the brain overgrowth. So we specifically looked at this protein called CD47. We see that in neural stem cells as well as oligodendrocyte progenitor cells derived from the autistic kids with brain enlargement, there is overexpression of CD47. That leads to reduced engulfment by the immune cells in the brain called microglia. We’ve found that if you block CD47 with blocking antibodies that have been developed for cancer, we can restore phagocytosis [ Ed. note : engulfment by immune cells] to normal levels.
What we ultimately hope to do is look at these pathways in other psychiatric disorders that are associated with changes in brain size, and also correlate our iPSC data back to brain imaging and behavioral data from the same individuals so that we have a deeper understanding of the relationship between the cellular phenotypes to the neuroimaging and clinical/behavioral measurements (a more personalized medicine approach). Our ultimate goal is to find more targeted therapeutics that could potentially help alleviate some of the symptoms in the kids who have autism by understanding these underlying mechanisms. One can potentially intervene at an early stage if the mechanisms are well-known.
RH: You mentioned that you work on schizophrenia too — is that related to brain growth?
SC: There is one psychiatric disorder called 16p11.2 duplication syndrome which is often associated with schizophrenia, where individuals can have a microcephalic, or a very small brain. We are planning to study these individuals as well, and see if similar mechanisms are underlying reduced brain growth. One idea that we have is that there is potentially too much elimination of cells, either through the complement system that activates the immune system or through changes in the balance between ‘eat me’ and ‘don’t eat me’ signals (such as CD47) as we’ve found to play an important role in the brain overgrowth models. In the long run, we will investigate if these imbalances in the neuroimmune system may be more generally involved in regulating brain size.
RH: How are you finding life as an assistant professor?
Chetty getting ice cream with her lab and daughter.
SC: It’s been a lot of fun, I really enjoy it. In the early years, it definitely takes some time to build up. In the first year, you’re waiting on new equipment to arrive, waiting for the right personnel to join the lab, and it takes some time to get your name out there so that students and postdocs know about you. But it definitely picks up within a year or two. The momentum has picked up a lot, and there are many different projects ongoing in the lab, which is a lot of fun. I also enjoy mentoring students and postdocs and seeing them do well. It’s really nice to attend and present our work at conferences, form new collaborations, and get feedback from others, and often my whole lab (including students, postdocs, and research technicians) gets the chance to go and present on their work as well. It takes time initially and you don’t know when you’ll reach that stage when it starts to just flow, but once you do reach it, after a year or two, it’s very nice to see the work getting published and rewarding to see your students and postdocs do well.
RH: Do you have any advice for prospective or current graduate students?
SC: I would say that choosing a lab where you’re really excited about the research questions is probably most important. You want to be in an environment where you’re excited to go into work every day, and keep working with the cellular or animal models, or whatever the techniques and the systems are in that lab. You want to be really passionate and excited to go in at any time to do the work. And ideally, it wouldn’t feel like work to you — it feels like you’re just having fun. The research questions have to be aligned with your own interests, and your long-term interests. I think that helps keep you motivated and keeps you asking new questions. Even after you’ve answered one, you will be eager to improve it and tackle the next question.
Finding good mentors and collaborators is very important as well. At Berkeley, Harvard, and Stanford, I’ve been really fortunate to have wonderful mentors and collaborations, both within the lab and outside the lab. With new collaborations, you have opportunities to learn new techniques and skill sets that are outside of your own space that may help you address a scientific question in a new way. I would say: seek good mentors and collaborators, and make sure you’re excited about the science that you are working on.
RH: What do you enjoy doing outside of work?
SC: My husband and I have a four-year-old daughter. Spending time with her is very enjoyable. It’s so fun to watch her grow, especially being in the neurodevelopmental field. When she was a baby, I could see all those things happening so quickly — the brain is developing so fast — and how important environmental enrichment can be for a young baby’s brain development. I like spending time with our daughter and family, being outdoors at parks or taking long walks, and exploring restaurants in the Bay Area.
A brain in a bottle, hmmm? And it’s helping a Stanford neuroscientist do his research?
Exaggerating, maybe. Kidding, no. And we’re not talking about computers. A Stanford neuroscientist is growing brainlike blobs in dishes, and they’re helping him learn a whole lot about his favorite subject.
3-D brain cultures are suspended in a lab dish at the lab of Sergiu Pasca, MD, assistant professor of psychiatry and behavioral sciences. (Photography by Timothy Archibald)
A brain is a complicated thing — the most complicated thing in the universe, some say — with close to 90 billion nerve cells, or neurons, and some 150 trillion individual neuron-to-neuron connections, called synapses. So, not such an easy entity for researchers to wrap their heads around.
Get out your 3-D glasses, because what follows reads like science fiction: Suppose you hope to learn what goes wrong during early brain development. One could learn a great deal about an individual’s neurodevelopmental condition by studying that person’s neurons close up, at the molecular, cellular and circuit levels. But how? You can’t exactly scoop a chunk out of someone’s living brain. And dead ones don’t tell you nearly enough.
Is there a workaround? A team led by Sergiu Pasca, MD, assistant professor of psychiatry and behavioral sciences, has found one — a technique that reliably and selectively produces pinhead-sized replicas of specific, different human brain parts in laboratory dishware. While researchers had previously developed other ways to culture cells to form brain organoids — minute clumps of tissue enriched for brain cells — these organoids also contained stray cells from other parts of the body, and they lacked the structural organization that characterizes components of a real brain.
Pasca’s method is notable because the clumps he’s growing contain only cells that are supposed to be in the brain, and because, rather amazingly, the clumps’ structures actually recapitulate those of distinct brain regions. His technical term for these little brain balls is brain-region-specific spheroids.
Pasca published his method for creating the brain balls in 2015 and knows of a dozen laboratories around the world that have successfully created them since then. Based on the hundreds of questions he has received about the protocol, he assumes there are many more.
The spheroids enable researchers to zero in on the pathological mechanisms that disrupt fetal brain development in autism, epilepsy and other neurodevelopmental disorders. They can also help neuroscientists understand the causes of faulty brain development in prematurely delivered babies.
“This is our doorway into personalized psychiatry,” says Pasca.
Sergiu Pasca uses this imaging system to observe the 3-D brain cultures he and his team create to study brain development. (Photography by Timothy Archibald.)
A brain ball is not a brain. The constructs are, at present, devoid of some important cell types found in a real human brain. They receive no sensory inputs from the outside world, and they can’t initiate muscular contractions. They also lack blood vessels, whose absence means a brain ball has to get its nutrients only at its surface, limiting its size.
Scientists are far from being able to grow a brain in a bottle. But as researchers learn to create more complex brain organoids and consider transplanting them into animals, ethical quandaries will multiply.
Pasca’s interest in research all started with chemistry experiments in his childhood home in Transylvania, a fabled region of Romania where he was born in 1982. He set up a chemistry lab in his parents’ basement at age 11 and promptly presented them with his first product: a crater in their backyard. His mastery of the subject improved, and in his final year in high school he won a national chemistry competition and a free ride to a nearby medical school. There, he met the woman who is now his wife, Anca, in a microbiology class. She was from Transylvania, too.
“I was born 30 miles from Dracula’s castle,” says Anca Pasca, MD, a clinical fellow in neonatology in Stanford’s Department of Pediatrics. Sergiu Pasca realized early in his medical training that his primary interest was research. But the school he was attending was so resource-poor that his biochemistry professor had to tap her own salary to fund his project: analyzing numerous substances in the blood of children with autism in search of a biochemical signature.
“I needed blood,” recalls Pasca, “so I would approach parents outside of a treatment center and ask them if I could get samples from their kids. You might expect the parents to be a little suspicious — after all, this was Transylvania — but instead these people would cry and hug me and thank me for working on this disease.”
Studying autism up close
Eventually, Pasca decided that “blood is pretty far from the brain.” He wanted to study autism up close, at the level of the neuron. And he knew exactly where, and with whom, he wanted to study it. He came to Stanford in 2009 to pursue a postdoctoral fellowship in the lab of Ricardo Dolmetsch, PhD, then an assistant professor of neurobiology and now global head of neuroscience at Novartis Institutes for Biomedical Research in Cambridge, Massachusetts. Dolmetsch had redirected his research to autism spectrum disorder after his son was diagnosed with it.
“Coming from an unknown medical school in Romania, without any molecular biology experience, and barely able to speak English, I thought I had no shot at joining such a successful lab,” Pasca says. “But Ricardo was looking for somebody with an interest in autism, and he gave me a chance.”
When Pasca started his postdoc in 2009, researchers were exploring ways to grow specific cell types from induced pluripotent stem cells, which had recently been discovered. Like embryonic stem cells, induced pluripotent stem cells (known as iPS cells) are capable of differentiating into virtually all the body’s different cell types. But unlike embryonic stem cells, iPS cells can be obtained, with relatively routine laboratory procedures, from any person’s skin. Dolmetsch wanted to generate neurons derived from the skin of a patient with a rare form of autism called Timothy syndrome that is caused by a genetic mutation. Pasca signed on.
In 2011, Dolmetsch and Pasca succeeded, which allowed them to pinpoint a mutation-induced physiological malfunction responsible for the symptoms of the disorder. Theirs was the first model of autism built from neurons that mirrored those in the brains of patients. The scientists hoped to monitor these neurons’ development over a longer term. “But the two-dimensional cultures we were using were too constrained,” says Pasca. “The cells didn’t act quite as they would in a human brain. Most 2-D cultures disintegrate after 100 days or so. But neurogenesis in the human cortex is complete only by 26 weeks of gestation, and astrocytes, brain cells that are absolutely essential for making working synapses between neurons, don’t even start getting made until late in gestation.”
Pasca resolved to improve the culture situation. That summer, he began noodling around with what he calls a “Saturday experiment” of trying to perfect a three-dimensional environment for culturing brain cells. By coating the bottom of the dish with a nontoxic repellent, he coerced neural progenitor cells to float freely in their nutrient broth, rather than hug the bottom of the dish as is their wont. Thus suspended, the cells proliferated, differentiated and clustered into tiny balls. These almost perfectly round clusters of cells continued to grow, differentiating further and eventually approaching one-sixth of an inch in diameter and perhaps a million cells apiece, forming brain-region-specific spheroids.
“The 3-D experiments were more of a game in the beginning,” Pasca says. “They started as an exploration of how much self-organization one can see in a dish after coaxing stem cells toward a neural fate. I was new to developmental neurobiology and fascinated by how cells assemble to form such complex structures as the mammalian central nervous system.”
Anca Pasca, who was doing her pediatrics residency at Stanford during that time, would often come to the lab to do experiments on her own. “After we moved here in 2009, Sergiu began spending a lot of time in the lab,” she says. “We had no friends and no family here, and I was getting bored staying home studying for my medical boards. We started wondering, what if we let these neural progenitor cells just float, suspended, in the dish instead of letting them attach?”
“She thought this could be a big thing,” says Sergiu Pasca, “and she continued to work on optimizing the culture medium and characterizing the little spheres.” Initially, Pasca and his colleagues produced brain balls by seeding their 3-D cultures with neural progenitor cells they generated from iPS cells grown in standard 2-D cultures. (Pasca now refers to this as the “2.5-D” method.) But eventually they learned how to proceed entirely in three dimensions, starting with actual iPS cells they then guided, in 3-D culture, through the neural-progenitor phase and then all the way to full-fledged, all-brain-and-only-brain brain-ball status.
Pasca’s brain balls can remain viable for up to two years or longer in culture — a record duration, long enough not only for the neurons they contain to mature thoroughly, but for their resident neural progenitor cells to spawn viable astrocytes, too. That, in turn, makes it possible for the neurons to form functioning synapses and complex circuits.
By mid-2014, Pasca had an assistant professorship in a tenure-track job and his own lab at Stanford. He also had multiple rounds of culture-medium optimization and brain ball characterization under his belt. The neurons in the brain balls acted, for all practical purposes, like the ones in a living brain. Their synapses were in working order, allowing neurons to form complex circuits through which they could talk to one another. These circuits closely approximated the real architecture of the brain region they’d been coaxed to mimic.
“I found myself wondering why people hadn’t tried this before,” says Pasca. In fact, others have used 3-D tactics to produce cultures enriched for brain cells, but they contained haphazard combinations of cells from other tissues. They also lacked the Pasca brain balls’ astounding brain-region mimicry.
Patience yields results
The key missing ingredient: patience. While others simply allowed their cell cultures to grow in a dish in a hands-off, nondirected fashion, the Pascas and their lab mates coaxed and coached their brain balls along desired developmental pathways by applying various combinations of small-molecule nutrients, trial-and-error style, iteration after iteration.
The Pasca group published its findings in a paper in Nature Methods in May 2015. One of the most amazing things about their brain balls was that, with not much chemical guidance, they tended to take on a default structure that’s a facsimile of the most evolutionarily advanced part of the brain: the human cerebral cortex, with all six layers you find in a living human brain.
The types and amounts of various proteins that each layer’s neurons were manufacturing mirrored those of the equivalent layer in an actual human cortex. And those neurons were alive and kicking: They sported ship-shape synapses, displayed spontaneous activity, fired in networked synchrony and were surrounded, as in real life, by their BFFs: happy, healthy astrocytes. (Astrocytes always pop up spontaneously in brain balls if you culture them long enough.)
“It’s amazing that these cells already self-organize and know what they need to do,” Pasca marvels. The iPS cells Pasca’s team uses as starter seeds for brain balls are generated from skin cells, so researchers can produce brain balls whose cells are genetically identical to the brain cells in the person whose skin is used. That makes brain balls powerful tools for studying any neurological disorder with a genetic component.
“Early on, I didn’t really believe this was going to work,” he says. “But Anca continued to help me optimize the protocols for the culture system until it did.”
Anca Pasca was a lead author of her husband’s 2015 paper, and he credits her for much of the hands-on work that helped to turn those “Saturday experiments” to a front-burner project. Her co-lead author, Steven Sloan, PhD, is an MD student who did his graduate work in the laboratory of the late Ben Barres, MD, PhD. On acquiring his PhD, Sloan has continued his research in the Pasca lab.
Throughout his postdoc period and beyond, Sergiu Pasca also was encouraged in his 3-D culture push by Barres, whom he calls his “second mentor.” Barres, who died in December 2017, was a professor of neurobiology, of developmental biology and of neurology and neurological sciences. Much of what’s known about astrocytes stems from Barres’ decades of research. The two notoriously late-night workers would engage in conversational collisions in the hallway or stop into each other’s adjacent offices when they saw the light on.
Later, when Pasca moved his lab to a new location, they would often exchange emails or text messages until early morning. “He really believed this could be done when nobody else did,” Pasca says. Barres also gave him an ultimatum: “He’d warn me that if my cultures weren’t producing astrocytes, they were crap.”
“Sergiu has accomplished something quite magnificent,” said Barres in an interview before his death. “The power and promise of this method is extraordinary. You can watch all kinds of brain diseases developing in a dish.”
The first Petri dish was put into service in 1887. Today, this most humble of scientific instruments remains at the cutting edge of discovery.
Molecular biologist Dr Madeline Lancaster was around 12 years old when she encountered her first Petri dish – one of her biochemist father’s postdocs happened to be growing some neurons. Her father took the dish, put it under the microscope and invited her to have a look. “I was just struck by the beauty and complexity of a neuron,” she remembers. “There is something wonderful about seeing something like that with your own eyes, and the way the light catches it. You can really see the dendritic tree, with all its tiny branches. The intricacy of it is beautiful. That was a real starting point for me.”
Petri dishes, invented by German microbiologist Julius Richard Petri while working for Robert Koch, rarely receive the accolades or the attention that their more complex lab companions like the microscope enjoy. And that’s understandable: they are simple, utilitarian little things, just shallow-lidded dishes, present in every child’s My First Chemistry Set. The real fascination of a Petri dish has always been what’s grown in it – and, indeed, it’s become a handy metaphor for a place where things grow. (A quick, non-scientific search brings up ‘Is Facebook the Petri Dish of Jealousy in Your Love Life?’ and ‘European Elections: A Petri Dish of Populist Dissent.’) When we think about the Petri dish’s most famous moment – Alexander Fleming’s discovery of penicillin – we marvel at the empty ring around the blob of mould, not the dish in which it sits. But Petri dishes deserve celebrating: they are still at the forefront of scientific discovery.
The invention of the Petri dish, and the advances it has helped to create, are part of a bigger whole, of course: the development of glass scientific instruments, from microscope lenses to laboratory beakers. “Glass shifts authority from the word, from the ear, the mind and writing, to external visual evidence,” says Alan Macfarlane, Emeritus Professor of Anthropological Science. In his book, The Glass Bathyscaphe: How Glass Changed the World (co-written with engineer, inventor and scientific instrument collector Gerry Martin), he argues that without glass, the Renaissance and the scientific revolution would never have happened. “Thus it could be argued that glass helped change the balance of power from the mind to the eye,” he says. “It makes glass a magical substance: a third kind of matter, neither fluid nor solid, in between.”
Around 70 per cent of what we know about the world comes in through our eyes, Macfarlane points out, and glass instruments enabled us to see better. “But until about 1400, most cultures were living intellectually on the past and what people had been told,” he says.“You didn’t look at the world, you listened. A child understood the world not by exploring it physically with sight, but by teachers telling them that the sun goes round the Earth or whatever. And the child just accepted it. Glass allowed the growth of the experimental method. Don’t trust what you are being told: see it for yourself. It was transformational.”
At the Wellcome-MRC Cambridge Stem Cell Institute, Professor Ludovic Vallier says that his first encounter with a Petri dish was a textbook example of understanding the world in this way: students used the dishes to see which bacteria could grow in the presence of antibiotics. “It’s good to see things grow,” he says. “It was a fascinating experience. Now, we grow cells in the Petri dish, and we don’t use glass any more, but plastic.”
“It’s rewarding to see something grow before your eyes. There’s something about the interplay between new, next-generation and classic technologies”
Today, his team focuses on human pluripotent stem cells which can be coaxed into becoming any cell type in the human body: neurons, skin cells, liver cells, and so on. Vallier and his colleagues study them in order to understand how they do this, and how they can produce more cells. And to study them, they need to grow them. “We put the stem cells on the dish and then we feed them and they grow, and they proliferate,” he says.
“And then, when they have become confident, we divide them and distribute them in new Petri dishes, and we grow them again. We feed them on a liquid medium that is basically food for cells: it tells them to grow and also what to do, as we want to produce new cells. So by feeding them this media we can allow the cells to become neurons, cardiac cells, liver cells, and so on. We can then model disease in a dish, or produce cells for regenerative medicine applications.”
Which means that the Petri dish becomes a place where Vallier and his team, jointly based at the MRC Cambridge Stem Cell Institute and the Wellcome Sanger Institute, can study – with everything from powerful microscopes to the naked eye – exactly what happens to those cells when disease strikes. “We work a lot on fatty liver diseases and in this case the liver cell in the dish becomes full of fat, which we can see. We can’t cut a piece of liver off a patient: we can’t look inside that liver to see what’s happening. So we are reproducing those diseases in our dishes, and screening new molecules to use against them.”
‘Disease in a dish’ is also the focus of Dr Meritxell Huch’s team at the Gurdon Institute. They use between 50 and 150 Petri dishes every day to grow primary mouse liver and human liver cells, in order to study how the liver can regenerate itself. After a week or so in the dish, surrounded by nutrients, they will self-organise and create a 3D structure called an organoid. Another one to two weeks will see the organoid become thick enough to break in two, creating another organoid that can be kept for more than a year in its dish.
Huch’s team is examining the molecular mechanisms by which these cells decide to proliferate. She says: “You can divide regeneration into different phases. The cells first have to realise that there is damage and activate the response. Once they activate the response, the cells will proliferate to compensate for the loss of cells owing to the damage. And once they have proliferated, they then have to become functional cells.”
Their research recently showed that they could take cells from a patient’s primary liver cancer and grow that same tumour in a Petri dish, reproducing its histology, architecture and genetic mutations. “This work has the potential to give us an answer to whether there will be a good or bad outcome for a given patient,” says Huch. “Our next step is to explore whether this could help us predict, or help to identify, drugs that could help a patient.”
This work has the potential to give us an answer to whether there will be a good or bad outcome for a given patient
Of course, the Petri dish itself has changed throughout the years. The majority are now plastic, not glass. They have been tweaked and improved to suit particular needs: Huch’s Petri dishes have a matrix made from the proteins collagen and laminin. Her cells don’t sit directly on the surface of the dish but are surrounded by this matrix, which supports stem-cell growth.
At her eponymous lab, based at the MRC Laboratory of Molecular Biology, Madeline Lancaster and her team grow ‘mini-brains’ in hundreds of Petri dishes. Here, the dish is used more as a vessel, specially treated to stop cells sticking and encourage them to float freely.
“We want the brain organoids to be three-dimensional rather than two-dimensional, as that’s the way our brains are,” she says. “If you can grow neurons on a dish in 2D, you can see individual neurons and see what they do, but you won’t be able to understand the architecture of those cells – their positioning relative to one another. You don’t end up with a good representation of how neurons are actually made in the brain. Neural stem cells, which are the stem cells that give rise to neurons, have a special orientation and they always make neurons in one direction. So if you put a bunch of those stem cells on a dish with no structural information, then they make neurons in random ways. Our method gives you a structure that looks a lot more like the structure of an actual developing brain.”
The aim of these organoids is to look at exactly how neurons are made and how that differs in humans compared with other species. One day, says Lancaster, this work could translate into understanding far more about Alzheimer’s disease, Parkinson’s and schizophrenia. So in a world of cutting-edge and highly complex technology, Petri dishes, in their relative simplicity, remain a vital tool in the fight against the world’s most difficult diseases. And, says Lancaster, they also enable a hands-on approach that she finds satisfying.
“It’s a bit like gardening,” she says. “You’re taking care of this thing. You keep an eye on it and you check it every day. You change the media this day or that day to help it grow better. It’s rewarding to see something grow before your eyes. There’s something about the interplay between new, next-generation and classic technologies. They give you capabilities that were just not possible before.”
Artist Klari Reis uses the tools and techniques of science in her creative process, collaborating with biomedical companies to create her unique work. The images on these pages are from her series A Daily Dish – a painting created in a Petri dish.