Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

Abstract

The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies.

Modeling human telencephalic development and autism-associated SHANK3 deficiency using organoids generated from single neural rosettes

“…Moreover, neural tissue with multiple rosettes-like structures has been observed in brain tumors [73][74][75] , which may confound the use of organoids for modeling normal development and disease. Second, SNR-derived organoids showed a relatively consistent and reproducible composition of different telencephalic cell types, which is similar to the results of other recent studies on organoids produced from single neural rosettes [76][77][78] . This allowed us to compare and contrast the cellular and molecular properties of control and isogenic SHANK3-deficient organoids to identify differentially expressed genes with no major differences in cellular composition.…”

A Shared Pathogenic Mechanism for Valproic Acid and SHROOM3 Knockout in a Brain Organoid Model of Neural Tube Defects

“…VPA, a well-known cause of NTDs in mice and humans, previously gave consistent expansion of the lumen area normalized to the entire SOSRS area at 200 and 400 µM doses 37 . Here we performed a larger dose-response curve from 50 to 800 µM using our newly established pipeline.…”

“…We have previously reported a protocol for generating self-organizing single rosette spheroids with manual imaging and analysis 37 . To dramatically increase throughput and decrease human bias, we have developed an entirely automated pipeline for imaging and analysis.…”

“…The SOSRS datasets are then filtered for individual lumens and size (7,800-31,415 µm 2 ). We use these filters to exclude SOSRS that result from the fusion of multiple monolayer fragments as shown in our previous publication 37 . To identify distinguishing features for particular treatments or genetic variants, we utilize the random forest predictive model followed by inspecting the individual feature importance.…”

“…Our group and others have published techniques for generating single rosette organoids or neural cyst cultures [34][35][36][37][38] . Our simple model of human neurulation, which we call SOSRS (self-organizing single-rosette spheroids), overcomes the lack of disease-relevant structural readout in previous brain organoid models.…”