PROJECT SUMMARY/ ABSTRACT The formation of neuronal laminae is a fundamental feature of cortical regions of the mammalian brain. This proposal aims to generate a defined 3D system of human cerebellar neurons that faithfully reproduces the native lamination of the cerebellar cortex. The cerebellum is a key area to study, as an archetypal cortical structure and as a brain region that functions both in sensory-motor control and in non-motor functions, including cognition, emotion and language. We will use two protocols in place in the Hatten Lab to differentiate the two primary classes of cerebellar neurons from human pluripotent stem cells (hPSCs) and state of the art bioengineering methods to develop a defined 3D laminar culture system (Aim 1). We will then use calcium imaging of cell-type specific genetically encoded calcium sensors to determine correlations in neuronal firing rates as a first step toward recording circuit properties of human cerebellar neurons in a 3D in vitro laminated system (Aim 2) and transcriptomic profiling against native developing tissue (Aim 3). Among cerebellar interneurons, unipolar brush cells are important modulators of the cerebellar circuit, yet no protocols are available to derive them in vitro. We will develop the first differentiation protocols to derive these glutamatergic neurons and incorporate them into laminar models (Aim 4). This research will combine the expertise of the Millen Lab in human cerebellar development with that of the Hatten Lab in generating human cerebellar neurons from hPSCs to provide the first properly stratified human cerebellar circuit for the purposes of disease modeling and drug screening.