Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here, we have adapted air-liquid interface culture to cerebral organoids leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies including long-range projection within and away from the organoid, growth cone turning, and decussation. Single-cell RNA-sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and extracortical projecting tracts can innervate mouse spinal cord and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.
Summary The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.
3D brain organoids are a powerful tool with prospective application for the study of neural development and disease. Here we describe the growth factor-free method of generating cerebral organoids from feeder-dependent or feeder-free human pluripotent stem cells using standard laboratory equipment. The protocol outlined below allows generation of 3D tissues, which replicate human early in vivo brain development up to the end of the first trimester, both in terms of morphology and gene expression pattern.
Three-dimensional neural organoids are emerging tools with the potential for improving our understanding of human brain development and neurological disorders. Recent advances in this field have demonstrated their capacity to model neurogenesis 1,2 , neuronal migration and positioning 3,4 , and even response to sensory input 5 . However, it remains to be seen whether these tissues can model axon guidance dynamics and the formation of complex connectivity with functional neuronal output. Here, we have established a longterm air-liquid interface culture paradigm that leads to improved neuronal survival and allows for imaging of axon guidance. Over time, these cultures spontaneously form thick axon tracts capable of projecting over long distances. Axon bundles display various morphological behaviors including intracortical projection within and across the organoid, growth cone turning, decussation, and projection away from the organoid. Single-cell RNAsequencing reveals the full repertoire of cortical neuronal identities, and retrograde labelling demonstrates these tract morphologies match the appropriate molecular identities, namely callosal and corticofugal neuron types. We show that these neurons are functionally mature, generate active networks within the organoid, and that extracortical projecting tracts innervate and activate mouse spinal cord-muscle explants. Muscle contractions can be evoked by stimulation of the organoid, while axotomy of the innervating tracts abolishes the muscle contraction response, demonstrating dependence on connection with the organoid. Overall, these results reveal a remarkable selforganization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and response to injury.
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