Quantitative profiling of human brain organoid cell diversity across four protocols and multiple cell lines

Human brain organoids are powerful in vitro models for brain development and disease. However, protocol and pluripotent cell line choices influence organoid variability and cell-type representation, complicating their use in biomedical research. Here, we systematically analyze the cellular and transcriptional landscape of brain organoids across multiple cell lines using four protocols aimed at recapitulating dorsal and ventral forebrain, midbrain, and striatum. We introduce the NEST-Score to evaluate cell-line- and protocol-driven differentiation propensities and comparisons to in vivo references. Thereby, we establish a set of protocols that together recreate the majority of cell types in the developing brain and provide a reference of cell-type recapitulation across cell lines and protocols. Additionally, we identify early gene expression signatures predicting protocol-driven organoid generation. We provide easy access to our data through a web explorer, creating a reference for brain organoid research and allowing straightforward protocol and cell-line performance validation.