BrainSTEM: A multi-resolution fetal brain atlas to assess the fidelity of human midbrain cultures

Midbrain dopaminergic neuron (mDA) differentiation protocols for Parkinson's disease (PD) modeling and cell replacement therapy lack comprehensive transcriptomic evaluations against in vivo references. To address this, we constructed an integrated human fetal whole brain atlas and a midbrain subatlas to use as standards of comparison. From the whole brain atlas, we observed distinct brain-region-specific gene expression in most neural cell types, emphasizing the need to first evaluate protocols at the whole brain level to identify midbrain cells. These cells are then mapped to the midbrain subatlas for more refined neuronal subtype annotation. Using this biologically-driven multi-resolution mapping strategy which we termed BrainSTEM (Brain Single-cell Two tiEr Mapping), we extensively surveyed publicly available single-cell datasets of human midbrain culture models. We confirmed the presence of multiple midbrain cell types (‘on-target’), but also a substantial proportion of cells associated with non-midbrain regions (‘off-target’). This leads to an overall 'inflation' of mDA presence across all published protocols. BrainSTEM offers a systematic and unbiased framework for understanding the current state of midbrain models, providing a foundation for guiding future improvement of midbrain differentiation protocols for PD studies.