Fused human forebrain spheroids, iDISCO 3-D reconstructionPASCA LABScientists know little about the early development of the human brain. As a result, they also have limited data about how human brain development might relate to neuropsychiatric disorders, such as autism and schizophrenia. Since 2013, however, scientists having been studying the developing human brain using neurons derived from human induced pluripotent stem cells (iPSCs), which are cultured in three dimensions into pea-size structures that mimic the full organ. Two studies published today (April 26) in Nature advance these research methods. In one paper, Harvard’s Paula Arlotta and colleagues described the development of organoids, or “mini brains.” In the other, Stanford’s Sergiu Pasca and colleagues used neural spheroids—balls of tissue containing more than a million neurons each—to study the interactions of two brain regions crucial to the development of the cerebral cortex.

“The major conclusion is the confirmation/validation that the human pluripotent stem cells are plastic enough to generate the diversity of cells necessary to recreate human, early stages of neurodevelopment in a dish,” Alysson Muotri, who studies neurological diseases using iPSCs at University of California, San Diego, but was not involved in either study, told The Scientist in an email. “Every neuroscientist working with early brain development will be excited by reading these articles.”

Researchers hope to use brain organoids and spheroids to study neurodevelopment and neuropsychiatric disorders; these mini brains have already been used to study Zika virus infection–linked microcephaly and autism spectrum disorders.

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Because many neuropsychiatric disorders are influenced by a person’s genetics, it is difficult to study these diseases in standard animal models. Instead, these types of diseases “must be modeled using the cells from the patient, because that’s the only way that you can get the genome of that patient,” Arlotta said. “This is really what justifies fundamentally the need for these human models.”

“These two studies complement each other really well,” University of Pennsylvania neuroscientist Guo-li Ming, who also was not involved in the work, wrote in an email. “The organoids generated are different in these two studies, with the Arlotta one using the whole-brain protocol and focusing on cellular diversity. The Pasca paper, on the other hand, [involves] generating brain region-specific organoids and try[ing] to put individual pieces together.”

Pasca’s team studied a stage in fetal brain development in which GABAergic, usually inhibitory interneurons in the deep forebrain migrate toward excitatory, glutamatergic neurons closer to the brain’s dorsal surface—a region that develops into the cerebral cortex and contains both neuronal types. To that end, the researchers differentiated iPSCs to generate both GABAergic and glutamatergic neural spheroids, fused the two spheroids in a tube, and observed their interactions.

As expected, the interneurons migrated, in a jumping, or “saltatory” manner, toward the glutamatergic neurons. Once there, “they change their morphology. . . . The dendrites that they have become more complex, but more importantly they start making connections with the glutamatergic cells,” Pasca told The Scientist. In spheroids derived from cells of patients with Timothy syndrome, a form of autism that results from a gain-of-function mutation in a calcium channel, this migration was aberrant; blocking the calcium channel with a drug restored normal migration.

“Pasca’s paper shows, for the first time, that assembling pieces of stem cell–derived human forebrain can be mimicked in vitro,” Muotri wrote. “This is important because several neurodevelopment disorders have defects on these early stages, but [until now] there were no in vitro models to study these processes.”

Arlotta’s team tweaked a previous protocol for developing mini brains so that they could survive for more than nine months—longer than had been achieved in previous studies. The brain organoids matured to the point that they started to develop mature features, such as dendritic spines, and began firing in synchronized patterns characteristic of neuronal networks, the researchers reported. The team used single-cell mRNA sequencing to characterize the cell types present in the brain organoids at both three months and six months, finding that, by six months, the organoids included seven different neuronal cell types, including retinal and cortical cells, and even more subtypes. Notably, the researchers found, the retinal cells responded to light.

“For the first time we have a system in which we use a normal, sort of semiphysiological sensory stimulus to stimulate neurons within the organoids,” Arlotta said. This is important, she said, because in studying neuropsychiatric disorders, researchers want to study brain organoids in settings as close as possible to human physiological conditions. “Rather than generating optogenetic channels, here we have cells that respond to normal sensory stimuli like light.”

F. Birey et al., “Assembly of functionally integrated human forebrain spheroids,” Nature, doi:10.1038/nature22330, 2017.

G. Quadrato et al., “Cell diversity and network dynamics in photosensitive human brain organoids,” Nature, doi:10.1038/nature22047, 2017.