The human induced pluripotent stem cell (hiPSC) technology promises major advances in disease modeling and personalized medicine. Using hiPSCs, organoid systems have been generated in recent years that resemble the identity of several brain regions, including cortex, basal ganglia, cerebellum and spinal cord. Major shortfalls of these models are the lack of a reproducible topography of the cell types and tissue architecture that are generated, and the failure to recapitulate the full range of cellular and molecular diversity that characterizes in vivo systems. Thus, our goal is to generate a more accurate and reproducible model for formation of human brain regions and their interactions in vitro. During early development, gradients of diffusible morphogens program cellular identity along the major dimensions of the vertebrate body, the antero- posterior (A-P) and dorso-ventral (D-V) axes, conveying positional information by inducing specific genetic programs. Recreating these morphogen gradients in vitro promises to increase the diversity of the organoid's cellular repertoire and its reproducibility. We focus on two signaling cues, WNT and Sonic Hedgehog (SHH), which, respectively, caudalize and ventralize the early neural tube in mammals. Naïve neural organoids tend to generate dorsal forebrain if not exposed to any patterning signals, and indeed cerebral cortical (CTX) fate is the default identity for the nervous tissue. In Aim1, we will use specially designed mesofluidic chambers to create stable concentration gradients of the posteriorizing morphogen WNT to generate organoid identities along the A-P axis (cortex-diencephalon-mesencephalon-brainstem) from 10 biologically different hiPSC lines. In parallel, we will test that hiPSC exposed to a concentration gradient of SHH will generate organoids identities along the D-V axis (hypothalamus- caudal- lateral-medial ganglionic eminences-cortex). Regional and cellular fates will be assessed by immunocytochemistry (ICC), single cell RNASeq and DBiT-seq, a novel spatial in situ transcriptomics approach. We will then test whether morphogen-induced initial specification achieved through the methodology proposed here will result in accurate and reproducible connections by developing multi-organoid aggregates (i.e., assembloids). In Aim 2, we will assemble region-specific organoids to form components of the cortico-basal ganglia-thalamo-cortical circuit. By labeling neurons with specific reporters, we will examine their projections to the adjacent regions and will test the functional activity and synaptic development of those projections using optogenetics. Generation of a series of differentially induced regions in close spatial proximity is important to allow subsequent migration and appropriate wiring of the CNS. Our approach promises to deliver a new system for modeling neuronal fate and circuitry development in humans and testing its functionality on the cellular, molecular and genomic level.

The induced pluripotent stem cell (iPSC) technology promises major advances in disease modeling and personalized medicine. The aims of this application are to use newly designed microfabricated, microfluidic chambers to produce iPSC-derived brain organoids reproducibly patterned in a series of brain regions topographically ordered in the anterior-posterior and dorso-ventral brain axes. We will also use new tools and analytical approaches to assemble these organoids into components of the cortico-basal ganglia-thalamo-cortical circuit, analyze their in situ gene expression, reciprocal connections and functional connectivity.