A great deal remains to be discovered about how the human brain develops into its mature form, starting with its origins in a tube-like structure that emerges early in embryonic development called the neural tube. From out of this humble tube, the brain and spinal cord grow. The nervous system’s process of self-assembly, guided by developmental programs evolved over millions of years and stored in our genes, is one of the great miracles of nature.

The very beginnings of the process are understood in part because they have been observed in animal models of brain development. While suggestive, these models diverge from the human developmental process in important details. In recent years researchers have developed specialized tools to create laboratory models of early human brain development, an achievement that is shedding light not only on how the human brain develops but also on problems and pathologies that perturb peripheral and central nervous system development and result in neurodevelopmental disorders that include autism and schizophrenia.

Now, in the journal Science, researchers describe their success in modeling a crucial phase of early human brain development for the first time. The research was directed by Sergiu P. Pasca, M.D., whose work has been supported by 2017 BBRF Independent Investigator and 2012 Young Investigator grants. Neal D. Amin, M.D., Ph.D., a 2021 BBRF Young Investigator, was co-first author on the new paper.

Dr. Pasca and his team are among the leading developers of new technologies that are revealing the secrets of human brain development. He was a pioneer in harnessing stem-cell science to create brain “organoids,” structures grown in a lab dish derived from patient blood- or skin-cell samples. Reprogrammed to develop as neurons or other brain cell types, cells in brain organoids wire together and begin to function. When transplanted into living rodents, they integrate with the functioning brain. This provides a window on normal brain development, but also on pathologies that perturb it, at the level of cells and circuits.

The new research relies on a technique Dr. Pasca perfected several years ago, to integrate organoids comprising different brain-cell and central nervous system cell types into larger, more complex entities he calls “assembloids.” The aim of the new assembloids created by the Pasca lab was to model the process through which neurodevelopmental guideposts called “organizers” orchestrate cell patterning and axon guidance in the spinal cord of the emerging nervous system.

Some of this biology has been modeled in mammals, leading to fundamental discoveries about how cells within a structure called the floor plate help direct neuronal axons to cross the spinal cord’s midline. The uniquely human aspects of this process remain mysterious, which led Dr. Pasca and colleagues to build assembloids comprised of three human cell-based organoid types to model the process as it unfolds in people.

The floor plate is a specialized group of cells at the ventral (i.e., bottom) midline of the developing neural tube. The floor plate is transitory group of specialized cells that secrete crucial patterning signals and can guide axons crossing from one side of the spinal cord to the other. This process of “crossing” establishes bilateral connectivity, which is critical for coordinating left-right movements. Problems that crop up during this early process of establishing midline connectivity have been implicated in neurodevelopmental disorders.

The Pasca team proceeded in several steps. First, they developed organoids that resembled the floor plate in humans. These were combined and integrated with separate organoids resembling the human spinal cord. The product of these multiple organoids, once combined, was an entity the team calls “midline assembloids.”

Various experiments established that the floor plate organoids faithfully generated patterning and axon guidance signals that are present in the human floor plate and are responsible for guiding axons across the midline and establishing connectivity across the spinal cord.

In an important set of experiments, the team analyzed the “secretome” of the cells comprising the floor-plate assembloids. The secretome is the complete set of molecules secreted by a cell into its surrounding environment, and the basis for interactions among cells of all kinds. The team found 27 genes whose expression profile was markedly different in humans from that seen in corresponding floor-plate cells in the developing mouse brain. These are clues to unique aspects of human brain development.

Using the gene-editing technology called CRISPR, the researchers deleted various of the human-enriched floor-plate genes, and observed the impact on axon guidance directed by floor-plate cells in midline assembloids. This revealed that two genes in particular, called GALNT2 and PLD3, when deleted, significantly impaired axon guidance. Implying that these two genes are critical in the process, it suggests potential sources of pathology relevant to axon guidance in early development. It’s just one example of the kind of insights that the “assembloid” approach can generate, since such experiments can never be performed in the human fetus.

The assembloid model reported in their paper “holds potential for additional applications” the team said. “Expanding beyond the spinal cord, assembloids can model midline function in other brain regions such as the corpus callosum, which are often disrupted in neurodevelopmental disorders.” The corpus callosum a large bundle of nerve fibers that connects the right and left hemispheres of the brain, allowing them to communicate and coordinate functions.

Broadly speaking, the team noted, “organizer organoids offer a versatile toolset to advance our knowledge of human cell specification, axon guidance, and evolution, which are crucial to understanding human neurodevelopment and the impact of neurodevelopmental disorders.”