SUMMARY The modular organization of the cerebral cortex is defined by anatomically and functionally segregated cortical columns, as well as layer-specific anatomical and functional connections that span multiple columns. Dysregulation of the developmental processes governing cortical formation can result in dysmorphic features that have been implicated in numerous neurological and psychiatric disorders. Understanding the basic principles of cortical development has largely relied on animal models but recent advances in 3D organoid cultures using human induced pluripotent stem cells (hiPSCs) have provided unprecedented opportunities to study the intrinsic properties of human neural stem cells and neural progenitors that give rise to highly organized structures in the central nervous system. To date, hiPSC-based cortical organoid models have captured the molecular and cellular dynamics in early stages of fetal human brain development but diffusion limits within the culture system have prevented modeling of later stages of human prenatal and perinatal development. To better model these later stages of human brain development that give rise to laminar and columnar organization, we have developed a sliced organoid culture platform that allows for continuous neurogenesis and the emergence of hallmark features of human cortical anatomy. In this project we will further characterize and validate this strategy (Aim 1) using single-cell RNA-sequencing, immunohistology, electrophysiology and electron microscopy. We will also fuse dorsal and ventral forebrain organoids to allow for the integration of constituent cell types in the cerebral cortex arising from distinct lineages. We will perform anatomical and functional mapping of the circuitry using virus-based trans-synaptic tracing, calcium imaging, and electrophysiology, as well as pharmacological and genetic perturbations to probe the functional implications of laminar (Aim 2) and columnar (Aim 3) organization. Finally, we will perform clonal lineage- tracing to test the hypothesis that functional cortical columns arise from distinct progenitors and radial migration of daughter cells (Aim 3). In sum, these experiments will lead to a human stem cell-based model to understand the human-specific molecular and cellular processes that govern cerebral cortex development and the emergence of functional and anatomical specificity in cortical modules.

NARRATIVE Cortical development in the mammalian brain culminates in highly organized functional modules with specific anatomical connections. This project aims to advance 3D cortical organoid modeling using human stem cells to model late-stage developmental processes governing the emergence of functionally and anatomically discrete laminar and columnar organization. A model to understand the human-specific cellular and molecular mechanisms that give rise to cortical architecture will help to generate insight into the etiology of neurological and psychiatric disorders with cortical pathology.