Platform to accurately recapitulate and perturb cortical development and morphogenesis in vitro

Abstract The application's broad objective is to recapitulate critical features of the developing human forebrain, including its patterning, morphology, and regional cellular organization. The application also seeks to develop a robust platform to perform high throughput penetrant and cell type-specific genetic perturbations to elucidate the mechanisms underlying human forebrain development. In doing so, it develops and integrates novel technologies from stem cell and developmental biology, molecular biology, bioengineering, single-cell genomics, and imaging. Given that model organisms inadequately mirror human brain development and disease, and ethical constraints limit direct human brain studies, it's imperative to develop in vitro models that accurately represent its microenvironment, organization, and function. Accurately reproducing the developing brain's attributes and the ability to perturb the genetic network at scale is crucial to decipher mechanisms underpinning human brain development and disease. In preliminary work, we established bioengineering methods to print and differentiate single-lumen cysts into forebrain-like tissue. We further demonstrated that coupling these cysts with signal coated generates spatial gradients of signals. With the ability to expose hundreds of organoids per experiment to distinct signaling gradients and using statistical analysis, we could rapidly learn how to generate correctly patterned complex tissues. Using these approaches, we will generate organoids with correctly integrated choroid plexus producing cerebrospinal fluid, an adjacent cortical hem, a more distal correctly layered cortex, and ventral inhibitory interneuron progenitors. The cortical hem and choroid plexus, adjacent to the developing cortical tissue, are essential for proper cortical development. We expect to be successful, given our preliminary results, and believe that the resulting platform to study human brain development will be broadly useful to the community. Further in preliminary work, we established bioengineering methods for targeted penetrant perturbation of individual organoids on a coverslip at a scale that was impossible previously because of the very high costs. This platform will allow us to perturb hundreds of genes per experiment, and we will test and demonstrate its capabilities by identifying human-specific mechanisms of forebrain neural tube patterning and closure. Uncovering mechanisms underlying the patterning and morphogenetic events leading to neural tube closure is essential for understanding human brain development. In sum, the proposal will establish a platform to mimic human brain development more accurately and further establish the ability to perform penetrant genetic perturbations, both at a scale not previously possible. Together these abilities will allow the community to better understand the development, morphogenesis, and mechanisms governing human brain development and disease.