Abstract
Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.
Highlights
The development, patterning, and homeostatic maintenance of the human brain is complex and while considerable insights into mechanisms driving these processes have been obtained from studies in model organisms, species-specific differences in brain development and function can make it challenging to apply results from animal models to humans
A key limitation for research applications is the considerable variability in shape/ architecture and cell type composition present in individual organoids. This characteristic makes it challenging to design experiments to address the effects of genetic variants, therapeutic candidates, and other perturbations on CO morphogenesis or function that allow statistically supported conclusions to be drawn
We describe a protocol to efficiently generate human dorsal forebrain organoids with phenotypically uniform morphologies that are reproducibly comprised of similar proportions of cell types across independent batches
Summary
The development, patterning, and homeostatic maintenance of the human brain is complex and while considerable insights into mechanisms driving these processes have been obtained from studies in model organisms, species-specific differences in brain development and function can make it challenging to apply results from animal models to humans. Understanding the molecular basis underlying normal development, disease progression, and therapeutic options for human brain-associated diseases, including cancer, requires human models. The ability to generate brain organoids derived from human pluripotent stem cells provides an unprecedented opportunity to study context-dependent human disease pathologies in an
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