NEWS & ANALYSIS

Lab Dishes Up Mini-Brains No bigger than apple seeds, the cell clusters are simply referred to as “cerebral organoids.” But that careful language in a paper in this week’s issue of Nature belies the excitement of many neuroscientists at what it reports: the growth from human embryonic stem cells of semiorganized knots of neural tissue that contain the rudiments of key parts of the human brain, including the hippocampus and prefrontal cortex. “I f ind it amazing,” says Wieland Huttner, a neuroscientist at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, who was not involved in the study. “It’s not real brain— that’s clear. But I’m positively surprised that so many features are reproduced.” Developmental geneticist Madeline Lancaster and her colleagues, who grew the organoids in a Vienna laboratory, have already shown that they can use the organoids to probe how normal human brain development goes awry in a genetic brain disorder. Left to their own devices in a lab dish, embryonic stem (ES) cells will differentiate into a menagerie of tissues: beating heart cells, neurons, even hair and teeth. The trick for scientists has been to harness that potential, coaxing the cells to grow into the kinds of tissues they want to study or use for a therapy. Developmental biologists know that neural tissue is a sort of default fate for differentiating embryonic cells, and researchers

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have been able to grow a variety of specific neural cell types from ES cells. But the level of cellular organization seen in this latest brain-in-a-dish paper is a significant step forward, says Magdalena Götz, who studies neurodevelopment at the Ludwig Maximilian University of Munich in Germany. Lancaster’s work took place in the lab of Jürgen Knoblich, a developmental geneticist at the Institute of Molecular Biotechnology of the Austrian Academy of Science in Vienna. It exploits what Knoblich calls the “absolutely enormous self-organizing capacity of developing human cells. If you just leave them alone and provide a medium that is supportive enough, they do things on their own.” It also builds on work by Yoshiki Sasai of the RIKEN Center for Developmental Biology in Kobe, Japan. In 2008, he and his colleagues reported that mouse and human ES cells in culture could spontaneously form cell layers that resemble the cortical layers in the brain. Lancaster, a postdoctoral researcher in Knoblich’s lab, was trying to culture early neural tissue to better understand how and when developing brain cells switch from proliferation—making more of themselves— to differentiation—making more mature cell types, which don’t continue to divide. Lancaster started with techniques developed by Sasai and others that shepherd dividing stem cells toward a neural fate, but she was

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CREDIT: MADELINE A. LANCASTER

N E U R O D E V E LO P M E N T

also intrigued, she says, by the “miniguts” that another research team had grown in droplets of Matrigel, a gelatinous protein mixture that can help cells grow in three dimensions. So she embedded clusters of stem cell–derived neural cells in a droplet of the material (see diagram). The Matrigel droplets freed the cell clusters to grow larger and develop more complex structures, without any further coaxing. To increase the availability of oxygen and other nutrients to the inner layers of the structures, Lancaster put the Matrigel droplets into a slowly rotating bioreactor, which gently shakes them. Within a few weeks, Lancaster says, she noticed darker pigmented patches on some of the cell clusters. On closer inspection, she recognized the rudiments of eye tissue, a sign that more complex structures might be forming. When she described the data at a lab meeting, Knoblich says, “I was completely blown away. I couldn’t sleep that night.” When lab members looked inside some of the cerebral organoids, they found structures that resemble the choroid plexus (the cavity in the brain that produces cerebrospinal fluid), the cerebral cortex (the brain’s outermost layer), and retinal tissue. More detailed staining showed evidence that after 16 days of development, the organoids had what resembles forebrain, midbrain, and hindbrain regions. The team also found molecular markers for a variety of more specialized regions—including the outer subventricular zone (OSVZ), a feature of human, but not mouse, brains. Organoids grown from mouse ES cells did not develop an OSVZ region. The resemblance to a real brain only goes so far. The organoids do not have any blood vessels, so cells at their core die. They reach their maximum size—about 3 millimeters in diameter—after 2 to 3 months, Lancaster says, and after 4 months they don’t develop any new cell types. However, the cell clusters can apparently survive indefinitely in the bioreactor; the oldest ones have been in culture for nearly a year, the researchers report. The cerebral organoids may shed light on human brain diseases that are difficult to study in mice or other animals. For

Downloaded from www.sciencemag.org on January 9, 2014

Inside view. A cross section of a cerebral organoid shows neural stem cells (red) and neurons (green).

NEWS Study human brain development

Human pluripotent stem cells

Cultivate embryoid bodies

Induce neuroectoderm

CREDIT: (SOURCE) ADAPTED FROM M. A. LANCASTER ET AL., NATURE 500 (29 AUGUST 2013) © NATURE PUBLISHING GROUP, (ILLUSTRATION) C. SMITH/SCIENCE

example, the scientists used the structures to study microcephaly, a neurodevelopmental disorder in which the head and brain end up much smaller than normal. Rather than starting with ES cells, they took cells from a person with a particular form of the disorder and “reprogrammed” them into so-called induced pluripotent stem (iPS) cells. Mice are a poor model for that form of microcephaly; the specific genetic mutation responsible results in mice with brains that are only slightly smaller than normal. In contrast, organoids derived from the patient’s iPS cells were shrunken, and Knoblich’s team

Grow in gel droplet

Spin cerebral organoids in bioreactor

found a clue to why. Certain precursor cells were maturing earlier than normal, bringing tissue growth to a halt prematurely. The organoids are probably not yet useful for studying more complex neurodevelopmental conditions such as autism or schizophrenia, because those conditions involve more mature cells and complex cell connections. Lancaster and Knoblich also note that each organoid develops distinctively, resulting in significant differences in composition and structure that make it hard to do controlled experiments. The researchers are working on ways to

Model neurodevelopmental disorders

Brain gain. The differentiation process from pluripotent cells to organoid takes about 3 weeks.

grow more consistent organoids—and to incorporate some sort of vascular system so that the cell clusters can grow bigger and presumably develop further. They hope that additional teams will take up and improve the method. Götz and others says they plan to do just that. “For studying the cerebral cortex, this is the best model so far,” she says. “People will use it, and time will tell how useful it is.” –GRETCHEN VOGEL

C E L L B I O LO G Y

NIH Effort Gambles on Mysterious Extracellular RNAs The versatility of RNA is legendary. Inside “unassailable” evidence that plant and nemthe cell it transfers genetic information, atode cells communicate through exRNA. acts as an enzyme, and regulates genes. In In humans and other mammals, exRNAs plants and nematodes, short RNA sequences abound in blood, tears, saliva, and every other also act like hormones, carrying messages body fluid. Among the molecules wending between cells. Now, a $17 million program through our bodies are messenger RNAs that of research grants, announced this month by carry the instructions for making proteins the U.S. National Institutes of Health (NIH), and microRNAs that fine-tune gene activaims to determine whether extracellular RNA ity. The extruded RNA is typically enclosed (exRNA) has a similar communication role in in membrane-bound capsules such as exopeople—and whether it can be harnessed for somes (Science, 24 June 2005, p. 1862) or diagnosis and treatment of human diseases. accompanied by protein or lipid bodyguards, “This is an effort to get which protect it from RNAa new field going,” says “This is an effort to get destroying enzymes. Christopher Austin, direcBut definitive data that a new field going.” tor of the National Censuch exRNAs influence ter for Advancing Transla—CHRISTOPHER AUSTIN, recipient cells have been NCATS missing for mammals. “It tional Sciences (NCATS), one of the five NIH cenhasn’t been convincingly ters and institutes participating in the 5-year shown that a small RNA can be active when it exRNA initiative. enters a different cell,” says molecular bioloConventional wisdom long held that the gist Michael McManus of the University of different varieties of RNA molecules toil California, San Francisco. mainly within the cells that make them. For Many of the 24 newly funded projects more than a decade, however, researchers are probing practical uses for exRNA. Some have seen signs that cells emit RNA mol- researchers will assess whether exRNAs can ecules that travel throughout an organism serve as biomarkers—molecular indicators and alter the activity of target cells. So far, that help doctors diagnose illnesses or idensays molecular biologist John Mattick of tify which people are susceptible. A group the Garvan Institute of Medical Research in led by cardiologist Jane Freedman of the UniSydney, Australia, scientists have accrued versity of Massachusetts Medical School in www.sciencemag.org

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Worcester, for instance, will sift blood samples from nearly 3000 participants in the famous Framingham Heart Study in hopes of learning whether certain exRNAs foretell cardiovascular disease. Even if they are just junk spit out by cells, the molecules may have some diagnostic value. Other projects will investigate whether exRNA can be harnessed to fight diseases such as cancer, Huntington’s disease, and multiple sclerosis. For example, cancer biologists Thomas Schmittgen and Mitch Phelps, of Ohio State University, Columbus, are working to engineer cells to manufacture exosomes that home in on the liver and deliver a specific microRNA that stymies the growth of tumor cells. McManus is teaming up with plant biologist Olivier Voinnet of the Swiss Federal Institute of Technology in Zurich and colleagues to try to settle the question of whether exRNAs have a biological role in mammals. Voinnet was one of the first researchers to uncover evidence for exRNA signaling in plants more than 15 years ago. No one has so far presented comparable data for humans, he says, but he and his colleagues are willing to be convinced that we use this RNA communication channel. “We are ‘positively skeptical’ about the whole issue,” Voinnet says.

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–MITCH LESLIE

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