Abstract The goal of this proposal is to develop robust in vitro human cell-derived microphysical systems which faithfully represent key features of the developing human neocortex in vivo. Our work addresses three key challenges that have limited the development of these systems to date: (1) Building robust and reproducible organoids at high throughput. To obtain meaningful, statistically significant results from genetic and non-genetic perturbations, it is necessary to develop organoid systems which are robust and can be reproducibly assayed in large numbers. (2) Determining in vivo relevance to human neocortex. The utility of organoid systems is defined by the degree to which they reproduce key aspects of human brain development that are not recapitulated by model organisms. (3) Monitoring and perturbing activity longitudinally in situ. To address fundamental questions about cerebral cortex development in either health or disease, it is necessary to capture and experimentally influence the trajectories of cellular activity across the three-dimensional volume of developing organoids through chronic recordings and perturbations. We overcome these challenges by merging three research teams whose expertise spans microfluidics and microelectromechanical systems, bioengineering and stem cell biology, computational and systems biology, and theoretical physics. We exploit novel technologies we have developed independently including: (1) microprinting, droplet encapsulation and microfluidic-based sorting methods to build and enrich for organoids with the selected cell types and geometry at high throughput, (2) in situ single cell RNA sequencing and computational mapping methods to determine the robustness of cell type composition and in vivo relevance against previously obtained in vivo fetal tissue data, and (3) 3D embedded soft microelectrode technology that grows and stretches with the developing tissue to chronically monitor and perturb electrical activity over the course of development. Here, we propose to integrate, employ, and build upon these inventions to further conduct basic research on a unique aspect of human brain development. The cerebral cortex is dramatically expanded and gyrated in humans versus other closely related species. Outer radial glial (oRG) progenitors have been implicated in this expansion. We have previously identified molecular markers that define these cell types, built and tested a reporter human embryonic stem cell line that drives GFP in these cell types, and developed a novel viral barcoded library that allows us to establish lineage relationships using single-cell sequencing. Here, we will determine the developmental potential of these human-specific oRG cells. Specifically, we will determine the contribution of the differentiated oRG progeny to cerebral cortex architecture, cell types, circuit connectivity, and developmental trajectory. The success of this proposal will result in a robust reproducible pipeline to build organoids that will be invaluable in the study of human neocortical development and disease.

Narrative The project seeks to develop and integrate technologies that make in vitro human stem cell-based organoids to study human brain development robust and relevant. To do so, it will develop micropatterning and microfluidics to build organoids, software to map data from these organoids to human fetal tissue to ascertain their relevance, stem cell lines that report on the generation of key human specific cell types in the organoids and novel 3D embedded electrode arrays to chronically assay how the developing neurons mature and form circuits. In the longer term, the technologies we develop will allow us to uncover mechanisms underlying human developmental diseases.