Immune reactions against proteins commonly used as molecular scissors might make CRISPR/Cas9 gene editing ineffective in people, a new study suggests.
About 79 percent of 34 blood donors tested had antibodies against the Cas9 protein from Staphylococcus aureus bacteria, Stanford University researchers report January 5 at bioRxiv.org. About 65 percent of donors had antibodies against the Cas9 protein from Streptococcus pyogenes.
Nearly half of 13 blood donors also had T cells that seek and destroy cells that make S. aureus Cas9 protein. The researchers did not detect any T cells that attack S. pyogenes Cas9, but the methods used to detect the cells may not be sensitive enough to find them, says study coauthor Kenneth Weinberg. Cas9 is the DNA-cutting enzyme that enables researchers to make precise edits in genes. Antibodies and T cells against the protein could cause the immune system to attack cells carrying it, making gene therapy ineffective.
The immune reactions may be a technical glitch that researchers will need to work around, but probably aren’t a safety concern as long as cells are edited in lab dishes rather than in the body, says Weinberg, a stem cell biologist and immunologist.
“We think we need to address this now … as we move toward clinical trials,” he says, but “this is probably going to turn out to be more of a hiccup than a brick wall.”
A new version of the periodic table showcases the predicted properties of 2-D metals, an obscure class of synthetic materials.
Arrayed in 1-atom-thick sheets, most of these 2-D metals have yet to be seen in the real world. So Janne Nevalaita and Pekka Koskinen, physicists at the University of Jyväskylä in Finland, simulated 2-D materials of 45 metallic elements, ranging from lithium to bismuth. For each sheet, the researchers measured the average chemical bond length, bond strength and the material’s compressibility, how difficult it is to squeeze the atoms closer together. The team then charted those features in the new periodic table. The new work, described in the Jan. 15 Physical Review B, could help researchers identify which 2-D metals are most promising for various applications, like spurring chemical reactions or sensing gases.
These metals are similar to previously studied 2-D materials, such as the supermaterial graphene (SN: 10/3/15, p. 7) and its cousin diamondene (SN: 9/2/17, p. 12). But whereas those materials were made up of covalent bonds — in which pairs of atoms share electrons — these 2-D metals are composed of metallic bonds, where electrons flow more freely among atoms. “It’s a whole new type of family of nanostructures,” Koskinen says. “Sky’s the limit, for what the applications could be.”
Like other superflat materials, some potential 2-D metals might exhibit exotic quantum qualities, such as 2-D magnetism or superconductivity, the ability to transmit electricity without resistance. Such properties may make those materials useful for quantum computing, says Joshua Robinson, a materials scientist at Penn State not involved in the work.
Nevalaita and Koskinen created three periodic tables that chart the properties of 2-D metals with atoms in triangular, square or honeycomb configurations. Using their trio of tables, the researchers discovered that the properties of 2-D metals were related to those of their 3-D counterparts. For instance, atoms of any given metal arranged in a triangular lattice typically had about 70 percent the bond strength of atoms in the 3-D version of that metal. Square and honeycomb lattices generally showed about 66 percent and 54 percent the bond strength of 3-D metals, respectively. The periodic tables revealed similar relationships between 2-D and 3-D metals in bond length and compressibility. These findings could allow researchers to get a quick profile of a 2-D metal that has never been created in the lab or in a computer simulation, just based on the well-known characteristics of its 3-D analog.
Nevalaita and Koskinen also compared the stability of 2-D metals whose atoms were arranged in the three different configurations. The researchers found that many 2-D metals were stable in triangular and honeycomb patterns, but not in squares. Future computer simulations could examine the electric and magnetic properties of these materials, Koskinen says. Knowing the stability and property profiles of 2-D metals could inform which materials scientists fabricate in the lab.
“This is the tip of the iceberg in the area of 2-D metals,” says Mauricio Terrones, a chemical physicist at Penn State not involved in the work.
Closing out a series in the NBA Playoffs is never easy, but the task may have gotten more difficult for the Lakers.
In the fourth quarter of a potential closeout game against the Warriors, Anthony Davis was forced to exit the game after taking an inadvertent shot to the head from Warriors center Kevon Looney.
What does this mean for Davis and the Lakers? Here is what we know about his injury and status moving forward. Anthony Davis injury update While battling for position on the inside, Davis took an inadvertent elbow from Looney to the side of his head. Davis was taken out of the game, brought back for evaluation, and eventually ruled out for Game 5 with what the Lakers ruled as a head injury.
Here's the play where Davis was injured as well as the aftermath that includes him receiving attention on the bench before being taken to the locker room. Turner Sports' Chris Haynes reported that Davis was being evaluated at Chase Center, with the possibility of a concussion looming as a potential diagnosis. Haynes added that Davis was brought to the locker room in a wheelchair following his evaluation.
In a later report, Haynes added that Davis "appears to have avoided a concussion and is doing better now," a positive sign for Davis and the Lakers.
Lakers head coach Darvin Ham told reporters that he spoke with Davis, who he said "seems to be doing really good already." Davis exited Game 5 with 21 points (on 10-of-18 shooting), nine rebounds and three assists in 32 minutes.
Will Anthony Davis play in Game 6? Davis' availability largely depends on the diagnosis of his injury and whether or not he is dealing with a concussion. If Davis is not diagnosed with a concussion, there is a clear path for him to return to the floor for Game 6.
Here is an excerpt from the NBA's Concussion Policy: There is just a one-day layoff between Games 5 and 6 of the Western Conference Semifinals series between the Lakers and Warriors.
Lakers vs. Warriors schedule Here is the schedule for the second-round series between Los Angeles and Golden State, with the final two games airing on the ESPN family of networks.
Fans in the U.S. can watch the NBA Playoffs on Sling TV, which is now offering HALF OFF your first month! Stream Sling Orange for $20 in your first month to catch all the games on TNT, ESPN & ABC. For games on NBA TV, subscribe to Sling Orange & Sports Extra for $27.50 in your first month. Local regional blackout restrictions apply. SIGN UP FOR SLING: English | Spanish
Date Game Time (ET) TV channel May 2 Lakers 117, Warriors 112 10 p.m. TNT May 4 Warriors 127, Lakers 100 9 p.m. ESPN May 6 Lakers 127, Warriors 97 8:30 p.m. ABC May 8 Lakers 104, Warriors 101 10 p.m. TNT May 10 Warriors 121, Lakers 106 10 p.m. TNT May 12 Game 6 TBD ESPN May 14 Game 7* TBD ABC
On July 31, 2012, Maggie de Grauw looked out the window of her flight back to New Zealand after a holiday in Samoa and glimpsed a mysterious mass floating below. That mass turned out to be a raft of lightweight pumice rock, the product of an erupting underwater volcano called Havre. The 2012 eruption turned out to be the largest of its kind in the last 100 years. And now, the pumice raft has become a crucial clue in revealing the eruption’s surprisingly complex nature. Although underwater eruptions happen all the time, scientists have only recorded such events since the 1990s, and pumice rafts can often float under the radar. Typically, researchers use depth sensors aboard ships to examine the crime scene of an underwater eruption.
But “what we found on the seafloor was almost entirely different from what we expected,” says Rebecca Carey, a volcanologist at the University of Tasmania in Australia. Havre challenges the reliability of the geologic record when it comes to big deep-sea eruptions.
In 2015, Carey and her colleagues set out to get a more detailed view of Havre’s big outburst than what ship-based sensors could reveal. The researchers deployed a robot to measure the depth of the 4-kilometer-wide caldera. Another robot, operated remotely from a ship, allowed the team to get a closer look at specific features in and around the caldera, and to take rock and water samples. A bit of satellite-image detective work revealed the size and path of the pumice raft, which formed no more than 21 1/2 hours after the eruption ended.
The robotic diving duo provided a high-resolution topographic map of the underwater posteruption landscape. The map shows a massive rupture, lava from 14 different vents ranging from 900 to 1,220 meters below the surface, chunks of pumice, landslide deposits and a blanket of ash. This diversity of volcanic material was unexpected, the researchers write January 10 in Science Advances. Although the Havre event was larger than the 1980 eruption of Mount St. Helens, a similar type of volcano that shot a huge column of debris into the air, the seafloor data weren’t indicative of such a large eruption. “When you shoot a lot of material up into water, there’s resistance,” Carey says. “So you expect to see a lot of it deposited on the seafloor.” But using an old seafloor map of Havre and satellite data, Carey and her colleagues calculated that more than 75 percent of the material produced by Havre ended up in the 400-square-kilometer pumice raft. That raft eventually broke apart and washed up on Australian and other South Pacific beaches. Volcanic gases might have pushed debris to the surface, Carey speculates, but it’s impossible to pinpoint a cause.
Many submarine eruptions go unnoticed, and few have been mapped in this manner. Frequently, researchers rely only on clues on the seafloor surface to determine an eruption’s size. And, if Carey’s team had just done that, the researchers would have never known the true size and nature of the eruption.
“That is a real eye-opener from this study,” says Bill Chadwick, a volcanologist at the National Oceanic and Atmospheric Administration’s Pacific Marine Environmental Laboratory in Newport, Ore. “What they found tells us a lot about how submarine eruptions behave differently than those on land.”
And if the Havre data are any guide, previous estimates of underwater eruption size may be off. “Now we know that the geological rock record is unfaithful to these very large magnitude powerful events,” Carey says.
Ready for sketch comedy she’s not. But a 14-year-old killer whale named Wikie has shown promise in mimicking strange sounds, such as a human “hello” — plus some rude noises.
Scientists recorded Wikie at her home in Marineland Aquarium in Antibes, France, imitating another killer whale’s loud “raspberry” sounds, as well as a trumpeting elephant and humans saying such words as “one, two, three.”
The orca’s efforts were overall “recognizable” as attempted copies, comparative psychologist José Zamorano Abramson of Complutense University of Madrid and colleagues report January 31 in Proceedings of the Royal Society B. Just how close Wikie’s imitations come to the originals depends on whether you’re emphasizing the rhythm or other aspects of sound, Abramson says.
Six people judged Wikie’s mimicry ability, and a computer program also rated her skills. She did better at some sounds, like blowing raspberries and saying “hello-hello,” than others, including saying “bye-bye.” Imitating human speech is especially challenging for killer whales. Instead of vocalizing by passing air through their throats, they sound off by forcing air through passageways in the upper parts of their heads. It’s “like speaking with the nose,” Abramson says.
The research supports the idea that imitation plays a role in how killer whales develop their elaborate dialects of bleating pulses. Cetaceans are rare among mammals in that, like humans, they learn how to make the sounds their species uses to communicate.
When it comes to the dimensions of spacetime, what you see may be what you get.
Using observations from the collision of two neutron stars that made headlines in 2017 (SN: 11/11/17, p. 6), scientists found no evidence of gravity leaking into hidden dimensions. The number of observed large spatial dimensions — kilometer-scale or bigger — is still limited to the three we know and love, the researchers report January 24 at arXiv.org.
Just as insects floating on a pond may be unaware of what’s above or below the water’s surface, our 3-D world might be part of a higher-dimensional universe that we can’t directly observe. However, says astrophysicist David Spergel of Princeton University, a coauthor of the new study, “gravity might be able to explore those other dimensions.” Such extra dimensions might explain some conundrums in physics, such as the existence of dark matter (an as-yet-unidentified source of mass in the universe) and dark energy (which causes the universe’s expansion rate to accelerate), says coauthor Daniel Holz, an astrophysicist at the University of Chicago. “That’s why people get excited about these modifications.”
To look for any hint of leaking gravity, scientists turned to the light and gravitational waves emitted in the neutron star smashup detected on August 17, 2017. The light allowed scientists to find the galaxy where the neutron stars merged. Spergel, Holz and colleagues showed that, given the galaxy’s distance from Earth, the strength of the gravitational waves was as expected. Extra dimensions weren’t stealing, and thus weakening, the observed ripples.
A variety of theories predict extra dimensions of spacetime into which gravity could leak, but the new result applies only to large extra dimensions, Spergel says. That’s because the gravitational waves detected from the neutron star collision have wavelengths of thousands of kilometers. Tiny extra dimensions, smaller than a fraction of a millimeter across, have also been proposed, but they wouldn’t affect such extended ripples. One theory, proposed in 2000 by a group of theoretical physicists including Georgi Dvali, predicts a type of large extra dimension. The effects of gravity leaking into such dimensions would be visible only over long distances — explaining why gravity on smaller scales, such as the size of the solar system, behaves as if there are three spatial dimensions.
Because the gravitational waves don’t seem to weaken on their trek to Earth, they must travel more than about 65 million light-years before leaking into any potential additional dimension, the researchers concluded in the new study.
But other theories of extra dimensions are unaffected by the result. String theory, which posits that particles are made up of infinitesimal vibrating strings, predicts tiny extra dimensions that are curled up on themselves. “We’re not in any way ruling out string theory,” Spergel says. Another variety of extra spacetime dimension, of potentially infinite size, was proposed by physicists Lisa Randall and Raman Sundrum in 1999 (SN: 9/26/09, p. 22). But such theories also would not be ruled out, because gravity can’t penetrate very far into that type of extra dimension.
Neutron star mergers are “a completely new laboratory of testing gravity,” says Dvali, of Ludwig-Maximilians-Universität in Munich, who was not involved with the research. “This is absolutely fascinating and fantastic.” But, Dvali notes, the type of extra dimension he proposed back in 2000 already seems unlikely on these scales. “I would say there is already an extremely strong constraint on leakage coming from cosmology.” No matter how far we peer out into space, the universe seems to follow the normal laws of gravity in three dimensions.
For now, the dimensions of space remain as simple as 1, 2, 3.
Human activities are driving phosphorus levels in the world’s lakes, rivers and other freshwater bodies to a critical point. The freshwater bodies on 38 percent of Earth’s land area (not including Antarctica) are overly enriched with phosphorus, leading to potentially toxic algal blooms and less available drinking water, researchers report January 24 in Water Resources Research.
Sewage, agriculture and other human sources add about 1.5 teragrams of phosphorus to freshwaters each year, the study estimates. That’s roughly equivalent to about four times the weight of the Empire State Building. The scientists tracked human phosphorus inputs from 2002 to 2010 from domestic, industrial and agricultural sources. Phosphorus in human waste was responsible for about 54 percent of the global load, while agricultural fertilizer use contributed about 38 percent. By country, China contributed 30 percent of the global total, India 8 percent and the United States 7 percent.
High-energy particle beams can reveal how 2-D thin sheets behave when the heat is cranked up.
Researchers have devised a way to track how these materials, such as the supermaterial graphene, expand or contract as temperatures rise (SN: 10/3/15, p. 7). This technique, described in the Feb. 2 Physical Review Letters, showed that 2-D semiconductors arranged in single-atom-thick sheets expand more like plastics than metals when heated. Better understanding the high-temp behaviors of these and other 2-D materials could help engineers design sturdy nano-sized electronics. Commonly used silicon-based electronics are “hitting a brick wall,” regarding how much smaller they can get, says Zlatan Aksamija, an electrical engineer at the University of Massachusetts Amherst not involved in the work. Materials made of ultrathin, 2-D films could be ideal for building the next generation of tinier devices.
But electronics warm up as electric current courses through them. If 2-D materials in a nanodevice expand or shrink at different rates from each other when heated, that could change the device’s electronic properties — such as how well it conducts electricity, says Antoine Reserbat-Plantey, a physicist at the Institute of Photonic Sciences in Barcelona not involved in the research. It’s crucial to know how the thin films react to higher temps.
The new method uses a scanning transmission electron microscope to bombard a film with a beam of high-energy particles. That particle beam stirs up electrons in the 2-D sheet, making the electrons swish back and forth through the material. The collective oscillation, called a plasmon, occurs at a frequency that depends on the material’s density, explains Matthew Mecklenburg, a physicist at the University of Southern California in Los Angeles who was not involved in the work.
The plasmon frequency affects how much energy the particles of the microscope beam lose as they streak through the 2-D material: the higher the frequency, the denser the material, and the more energy that is sapped from the beam. By using another instrument to measure the energies of beam particles after they’ve passed through the 2-D material, researchers can discern the material’s density — and track how that density changes as they turn up the heat. Robert Klie, a physicist at the University of Illinois at Chicago, and colleagues used this technique on samples of graphene, which is made of carbon atoms, and four 2-D semiconductors made of transition metal and chalcogen atoms. (Chalcogen elements are found in group 16 on the periodic table and include sulfur and selenium). These materials were arranged in sheets from a single atom to a few atoms thick. The team measured the density of each sample at eight temperatures between about 100° and 450° Celsius. That allowed the scientists to calculate how much each material expanded or contracted per degree of temperature increase.
These measurements revealed that the thinnest structures undergo more significant size changes than thicker sheets: A single layer of graphene, which contracts when heated, shrinks more than materials composed of a few graphene layers. The 2-D semiconductors expand at higher temps, but those made of one-atom-thick sheets swell more than semiconductors a few atoms thick. In fact, the heat response of the single-atom-thick semiconductors is “more like [that] of a plastic than a metal,” Mecklenburg says.
This finding may indicate that, like plastics, some 2-D semiconductors have low melting temperatures, which could affect how or whether they’re used in future electronics.
Harbor porpoises are frequently exposed to sounds from shipping vessels that register at around 100 decibels, about as loud as a lawnmower, scientists report February 14 in Proceedings of the Royal Society B. Sounds this loud can cause porpoises to stop echolocation, which they use to catch food.
While high-frequency submarine sonar has been found to harm whales (SN: 4/23/11, p. 16), low-frequency noise from shipping vessels is responsible for most human-made noise in the ocean, the researchers say. Porpoises have poor hearing in lower frequencies, so it was unclear if they were affected.
In the first study to assess the effects of shipping vessel noise on porpoises, researchers tagged seven harbor porpoises off the coast of Denmark with sensors that tracked the animals’ movement and echolocation usage in response to underwater noise over about 20 hours.
One ship created a 130 decibel noise — twice as loud as a chainsaw — that caused a porpoise to flee at top speed. These initial results indicate that ship noise could affect how much food porpoises hunt and consume.
In a white lab coat and blue latex gloves, Neda Vishlaghi peers through a light microscope at six milky-white blobs. Each is about the size of a couscous grain, bathed in the pale orange broth of a petri dish. With tweezers in one hand and surgical scissors in the other, she deftly snips one tiny clump in half.
When growing human brains, sometimes you need to do some pruning.
The blobs are 8-week-old bits of brainlike tissue. While they wouldn’t be mistaken for Lilliputian-sized brains, some of their fine-grained features bear a remarkable resemblance to the human cerebral cortex, home to our memories, decision making and other high-level cognitive powers.
Vishlaghi created these “minibrains” at the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, where she’s a research assistant. First she immersed batches of human pluripotent stem cells — which can morph into any cell type in the body — in a special mix of chemicals. The free-floating cells multiplied and coalesced into itty-bitty balls of neural tissue. Nurtured with meticulously timed doses of growth-supporting ingredients, the cell clumps were eventually transferred to petri dishes of broth laced with Matrigel, a gelatin-like matrix of proteins.
On day 56, the blobs display shadowy clusters of neural “rosettes.” Under a laser scanning microscope, razor-thin slices of those rosettes reveal loose-knit layers of a variety of dividing neural stem cells and the nerve cells, or neurons, they give rise to. The layered structures look similar to the architecture of a human fetal brain at 14 weeks of gestation. Across the globe, labs such as this one, led by UCLA developmental biologist and neuroscientist Bennett Novitch, are cultivating thousands of these brainy clumps for research. Less than five years ago, a team of biologists in Austria and the United Kingdom and one in Japan wowed the world when they announced they had made rudimentary bits of 3-D human cerebral cortex in a dish. Since then, researchers have been eagerly tinkering with techniques for producing these miniature brain models, like chefs obsessively refining their favorite recipes.
“It’s like making a cake: You have many different ways in which you can do it,” says Novitch, who prefers using the Japanese method with a few tweaks. “There are all sorts of little tricks that people have come up with to overcome some of the common challenges.”
For instance, because the brain blobs lack a built-in blood supply, they must absorb enough oxygen and nutrients from the tissue-culture broth to remain healthy. To help, some labs circulate the broth around the tissue clumps. The UCLA researchers choose instead to grow theirs at higher oxygen levels and chop the blobs at the 35-day mark, when they are as wide as three millimeters, and then about every two weeks after. Sounds radical, but the slicing gives cells on the inside — some of which start dying — exposure to much-needed oxygen and nutrients. Those divided bits then continue growing separately. But cutting can be done only so many times before the expanding rosette structures inside are damaged.
With all the experimenting, researchers have cooked up a lot of innovations, including some nifty progress reported in just the last year. Scientists have concocted tiny versions of several brain regions ranging from the hypothalamus, which regulates body temperature, thirst and hunger, to the movement-controlling basal ganglia. Electrical chatter among neurons, reflecting active brain circuits, has been detected. And research groups have recently begun linking bits of specific regions like Legos. Scientists have even observed some early developmental processes as they happen within the human brain blobs. Stem cell payoff The work is part of a broader scientific bonanza that comes from coaxing human stem cells to self-assemble into balls of organlike tissue, known as organoids, that are usually no bigger than a lentil. Although the organoids don’t grow enough to replicate entire human organs, these mini-versions can mimic the 3-D cellular infrastructure of everything from our guts to our lungs. That’s something you can’t get from studies of rodents, which have different biology than humans do.
Mini-organ models promise enormous advantages for understanding basic human biology, teasing apart human disease processes, and offering an accurate testing ground for finding or vetting drug therapies. And by creating personalized organoids from the reprogrammed cells of patients, scientists could study disease in a very individualized way — or maybe even use organoid structures to replace certain damaged tissues, such as in the liver or spinal cord.
“Organoids offer an unprecedented level of access into the inner workings of the human brain,” Novitch says, noting that our brains are largely off-limits to poking and cutting into for research. If scientists can study accurate models of working neural circuits in these brain bits, he and others say, researchers might finally get a handle on uniquely human neurological conditions. Such disorders, which include epilepsy and, experts theorize, schizophrenia and autism (SN Online: 7/17/15), can arise when the brain’s communication networks develop off-kilter. But the research is still in its early days. Although there’s been exciting headway, studies sometimes overstate the extent to which human brain organoids reproduce features of actual developing brain tissue, says stem cell biologist Arnold Kriegstein of the University of California, San Francisco. The minimodels still lack many basic components, including certain cell types, a blood-vessel network and inputs from other neural regions.
Another stumbling block is that brain organoids can vary a lot from protocol to protocol, or even batch to batch within the same lab. “The major focus now needs to be on reproducibility, and being able to get an approach that you can rely on to give you the same outcome each time,” Kriegstein says.
DIY organs For decades, biology research has relied on cell lines grown in flat sheets in petri dishes, but those sheets lack the structural complexity of living tissue. Then came pioneering work that unveiled the do-it-yourself magic of stem cells raised free-floating in broth.
Organlike tissue bits can be generated from pluripotent stem cells that are either plucked from embryos or created by taking a person’s adult skin or blood cells and chemically inducing them to revert to an embryonic-like state. Starting in the mid-2000s, Yoshiki Sasai’s team at the RIKEN Center for Developmental Biology in Kobe, Japan, demonstrated how to grow brainlike structures using embryonic stem cells, first from mice and then humans. In their groundbreaking study in 2013 in Proceedings of the National Academy of Sciences, the researchers used chemical cues to direct human embryonic stem cells to form a specific region of the human cortex. (Tragically, Sasai committed suicide the next year, after two stem cell studies that he coauthored were retracted amid scientific misconduct charges against a research colleague [SN: 12/27/14, p. 25]. Before his death, Sasai was cleared of any direct involvement. The discredited studies were not related to the organoid research.)
A few months before the 2013 Sasai team paper, Madeline Lancaster and Juergen Knoblich of the Institute of Molecular Biotechnology in Vienna and U.K. colleagues demonstrated their more freewheeling, landmark approach to growing brain organoids (SN: 9/21/13, p. 5). The recipe, described in Nature, allows human pluripotent stem cells to spontaneously attempt to assemble into a tiny approximation of a whole brain by making whatever brain structures the stem cells choose.
Meanwhile, biologists elsewhere were whipping up other types of organoids, starting instead with adult stem cells. These rare, damage-repairing cells are found in many organs (including the brain), but the cells can transform into only a limited range of cell types. In 2009, Hans Clevers of the Hubrecht Institute in Utrecht, the Netherlands, announced that his lab unexpectedly created a miniature version of a gut while cultivating adult stem cells that the team had discovered in mouse intestinal tissue. Grown in a drop of Matrigel with a trio of growth-inducing factors, these cells coalesced into little spheres containing tiny projections that resembled the fingerlike villi that absorb nutrients in the gut.
Scientists soon were concocting tiny facsimiles of human stomachs, livers, kidneys, lungs and more (SN: 12/28/13, p. 20). “We essentially are discovering the vitality of what the stem cells actually do,” says Clevers, who is president of the International Society for Stem Cell Research. “We give [the cells] a little push, and they do whatever they’re good at.”
The trick is knowing exactly which ingredients to use to make different organs. For pluripotent stem cells, that means exposing them to just the right growth factors or inhibitors at just the right times, over about a month, says James Wells of the Center for Stem Cell and Organoid Medicine at Cincinnati Children’s Hospital Medical Center. Some of those essential instructions are well-known from decades of research on embryo development in fish, chickens and rodents; the same chemical cues generally work for all animals with spinal cords, including people. However, for many body parts, organoid makers must suss out recipe instructions from scratch. Working with Jorge Múnera and other colleagues, Wells recently produced a minimodel of a human colon using human induced pluripotent stem cells. But first, the team conducted months of experiments on frog and mouse embryos to identify the signals for forming a colon. “It took a while to figure out what the special sauce was,” Wells says.
Some scientists have distant dreams of using organoid methods to grow full-size livers or kidneys in the lab for transplantation. A more attainable goal may be regenerative tissue transplants, for example, replacing dying liver cells in someone with early-stage liver disease with chunks of healthy stem cells from a personalized liver organoid. Or, in patients who’ve had part of the small intestine removed, tiny pieces of gut organoid tissue could be implanted and, after growing larger, connected to the intestine.
Head games The human brain, meanwhile, is vastly more complicated than any other organ. It’s unlikely that scientists will ever be able to build a full replica. While the initial brain-making recipes were stunning for what they could achieve, they left much room for improvement. In the years since the 2013 debut of human brain organoids, research groups have worked to grow bigger brain tissue clumps and more uniform structures.
The Austrian method for making whole-brain organoids, in particular, produced a random mix of neural regions laid out in a topsy-turvy manner. But bioengineering tricks may help. In a study last year, Lancaster, now at the MRC Laboratory of Molecular Biology in Cambridge, England, and Knoblich got more consistent results by adding polymer filaments as scaffolding to guide the organization of the minibrain models. Other scientists, following the Japanese approach, which generally gives more predictable results, have concentrated on coaxing out specific cell types or structural features of the real brain. For instance, one constraint is that the organoids form slowly, more or less sticking to the same timeline of development as does a human brain during gestation. But without a blood supply, growth is limited; the brain bits reach only a few millimeters in size. That means organoid models are often short on cell types from later development stages, such as cells called astrocytes. These star-shaped cells are crucial for creating and curating the connections between neurons, and also may help with forming memories (SN Online: 11/15/17).
Astrocytes don’t fully mature in a baby’s brain until after birth. But Stanford University neuroscientist Sergiu Paşca has crafted a method for making and maintaining 4-millimeter-wide balls of human cortex–like tissue (he calls them spheroids) in 3-D culture for an extended time. Last August in Neuron, his team described organoids that survived for more than 20 months — long enough, analyses showed, for astrocytes to mature and function in ways that mimic their real-brain counterparts.
Of great interest, also, are the outer radial glial (oRG) cells, neural stem cells that are pivotal for constructing the unusually big cortex that’s unique to humans; oRG cells are scarce in mouse brains. When Novitch’s lab group at UCLA tried the original Japanese and Austrian organoid-making recipes, the output of oRG cells was underwhelming. So Novitch worked with Vishlaghi and postdoctoral researcher Momoko Watanabe to refine the protocol to pump up the cells’ production and reliably generate better cerebral blobs.
Among other tweaks, Novitch’s team added a dash of a molecule dubbed LIF, which recent studies by others had suggested can spur the oRGs to multiply. It worked, leading to a threefold increase in the oRG populations and enhanced growth of upper neuron layers. The researchers shared their revised protocol last October in Cell Reports. On a different front, labs have begun assembling more complex minibrain models, like playing with self-directed Legos. For two months, Paşca’s team at Stanford grew spheroids in separate sets of dishes that mimicked either cortex tissue or an adjacent underlying region known as the subpallium. Then the researchers put the different bits side-by-side and left them overnight in a culture tube. Similar to how the two regions normally connect in the developing brain, the little pieces knew what to do. “By the next day they are essentially fused to each other,” says Paşca, who announced the results in May in Nature.
During the fusion process, the researchers took time-lapse videos of long, spaghetti-like cells called interneurons migrating from a spheroid of the subpallium into a cortexlike spheroid.
“They don’t crawl, they actually jump,” Paşca says. The images capture aspects of a hallmark phenomenon that normally unfolds during the second and third trimester of fetal gestation.
Testing ground Once on the other side, interneurons form a circuit with — and quell the activity of — excitatory neurons in the cortexlike tissue, electrophysiological tests suggest. If not quieted, excitatory neurons will trigger neighboring cells to fire. In the real brain, maintaining a proper balance in neural network activity is important; disruptions in it appear to foster disorders such as epilepsy, and perhaps schizophrenia and autism.
Indeed, in the same paper, the Stanford team reported new discoveries using personalized brain spheroids derived from induced pluripotent stem cells of patients with Timothy syndrome — a rare condition caused by an overactive calcium channel found mainly in the brain and heart. Patients with the disorder have epilepsy, autism and heart problems. In the patients’ spheroids, interneurons migrated inefficiently but, by adding drugs that blocked the dysfunctional calcium channel, the researchers could reverse the problem. The brain organoids made these intriguing observations possible, Paşca says. “We couldn’t have done this in any other way.” Organoid experiments by others have, meanwhile, helped confirm that the Zika virus targets and kills oRG cells and other neural precursor cells, contributing to small brain size in infected infants.
In a 2016 study, Johns Hopkins University neuroscientists Guo-li Ming and Hongjun Song reported on their own techniques for creating brain bits that have a well-defined zone of oRG cells. After infecting these organoids with the Zika virus, the researchers observed a collapse of cortexlike tissue that may partly explain the stunted brain growth (SN: 4/2/16, p. 26). 2-D cell-culture and mouse experiments also provided key evidence of the virus’s modus operandi; although the rodent brain doesn’t harbor the full contingent of human neural stem cells, it has blood vessels and immune-system components that organoids lack.
In search of Zika-fighting treatments, Ming and Song, both now at University of Pennsylvania, and their colleagues have been screening thousands of compounds in 2-D cell cultures, and then validating the most promising candidates with tests in 3-D brain organoids. The team has found several potential antiviral and neuron-protecting agents to pursue. Novitch’s UCLA lab group has likewise used its brain organoids to pinpoint additional receptors by which the virus may gain entry into neural stem cells, and identified a few other drug leads for blocking infection.
Organoids may also prove valuable for tailoring treatments for patients, says David Panchision, chief of the developmental neurobiology program at the National Institute of Mental Health in Bethesda, Md. Researchers might generate personalized brain organoids from the reprogrammed skin cells of individuals with, say, schizophrenia and test which medications work best for patients with particular genetic profiles of the illness.
In the Netherlands, based on research reported in 2016 in Science Translational Medicine, Clevers and colleagues are already using personalized gut organoids, derived from rectal biopsies, to test whether cystic fibrosis patients will benefit from available drugs. Tailored regenerative therapies with 3-D substructures of neural tissue may also be possible, Panchision adds, for conditions like Parkinson’s disease or spinal cord injury. Growing pains For now, though, scientists have hefty challenges to overcome. Much work remains in optimizing how faithfully the bits of tissue reproduce normal brain function and architecture, Panchision says. For one thing, the organoids are developmentally young and don’t reflect a mature brain. And researchers must figure out how to build in some core features: the necessary blood vessels, immune-system cells called microglia and connections from other brain regions, such as the thalamus and cerebellum. Not to mention steroid and thyroid hormones, which also shape brain growth.
However, scientists don’t necessarily need or want to create a comprehensive replica of the human brain in a dish, Panchision and others point out. Rather, the goal is to build robust and reliable models for studying specific aspects of brain function.
Thus the pressing need for standardized, reproducible organoid-making recipes. Novitch’s group and many other labs are still trying to figure out why the brain bits can vary so much in size, composition and structure. Part of the trouble is the ingredients: Subtle variations in tissue-culture chemicals and Matrigel, or in different stem cell lines and how they are grown first in 2-D culture, can have a big impact on how the organoids turn out, Novitch says.
At the same time, researchers need to do a more thorough job of analyzing brain organoids to know what’s actually in them at different developmental time points, compared with actual human fetal brain tissue, says UCSF’s Kriegstein. It’s otherwise hard to say whether a brain blob truly recapitulates the neural tissue that scientists claim it does. Labs have started tackling the problem with a tool called single-cell transcriptome analysis, which gives readouts of all the genes that are active in individual cells.
“Greater rigor is needed,” Kriegstein says. “And I am sure we will eventually get there.”
