Project Summary Understanding of many aspects of the human brain is currently limited due to the extreme levels of complexity involved, the inability of animal models to adequately model human brain biology and function, and the ethical and practical limitations of working with human brain tissue in or from patients. Leveraging the ability of induced pluripotent stem cells (iPSCs) to self-organize into structures mimicking, at some level, the human brain, researchers have begun to develop so-called human brain organoids as in vitro models to enable the next generation of brain development and pathology studies. As human brain organoid technology evolves and structures, hierarchies, and behavior observed in this system more closely replicate that observed in vivo, there is increasing potential to provide critical insight needed to understand and treat currently incurable neurological disorders. Current approaches to engineer higher levels of topographic organization in human brain organoids, however, are still quite limited, with fusion of two organoids of disparate regional specification being the most common approach. This approach does not rely on the soluble signaling factor gradients that direct polarized organization, and results in structures that do not exhibit the complex spectrum of subdivisions observed in the brain. Recent work employing microfluidic devices has demonstrated that it is possible to establish time- varying, quantitatively predictable gradients of morphogens in small hydrogel slabs and drive embedded stem cells to a spectrum of differentiation states; these platforms, however, are far too small to work with human brain organoids. Inspired by these devices, we have developed a platform using fluidic channels within a large hydrogel to establish gradients of soluble factors in a large volume. We have demonstrated that computational modeling of the diffusion of morphogens in this device yields values that are in close agreement with experimentally measured concentration gradients, and employed the platform to drive embedded stem cells to a spectrum of differentiation states. The spatiotemporal control over soluble factor concentration provided by this platform, combined with its ability to work with large volumes, renders it uniquely suited to establishing a well-defined niche for a human brain organoid. Thus, we propose to use this fluidic hydrogel platform to establish gradients of signaling factors to direct an embedded human brain organoid to organize along dorsoventral (Aim 1) or anteroposterior (Aim 2) axes. By demonstrating a quantitatively tunable, reproducible process that can be easily used with any set of soluble signaling factors, the results of this work will pave the way for the next generation of human brain organoid research, enabling higher degrees of complexity and more biomimetic organization to facilitate new insight into human brain development and pathology, and to help develop new treatments for neurological disorders.