PROJECT SUMMARY Understanding the principles that govern the development of the enormously complex human brain is a fundamental goal in biology. Human pluripotent stem cells have proven to be a powerful tool in this effort as an active field of researchers have leveraged this technology to define the factors required to trigger endogenous genetic programs that ultimately give rise to diverse specialized brain cell types. One of the most exciting recent applications of this technology is the generation of tri-dimensional (3D) brain cell cultures or "organoids," which enable neural progenitors to proliferate, differentiate, and self-organize into complex microphysiological systems. Brain organoids have been shown to develop structural, transcriptional, and functional similarities up to the mid-to-late gestation human brain with remarkably little external input. Currently they represent the closest cellular model to native human brain tissue available. While an enormously powerful system for probing mechanisms of prenatal development, efforts to access postnatal stages with brain organoids have been frustrating. Numerous attempts to improve upon the maturation state of brain organoids have proved to be incremental, inconclusive, or poorly reproduced. As a result, brain organoid systems remain largely inappropriate for modeling advanced stages of development (e.g., postnatal brain). At the same time, these prior studies underscore the momentum and unmet need to improve models of the human brain. Given the potential for fundamental and clinical advances, breaking this developmental wall should be considered a top priority in neuroscience research. My core hypothesis is that a major reason brain organoids fail to develop past late gestational time points is because they lack developmental guidance from significant sources of inputs. The primary objective of this proposal is to unlock advanced stages of human brain organoid development by applying a multipronged approach to replace missing exogenous neural inputs in brain organoids with synthetic "virtual" inputs and to determine the effects such manipulations have on development, with an emphasis on the specification of diverse neuronal cell types and acquisition of advanced states of activity. We will try several methods in parallel that activate cells and circuits over extended periods of time and in different biologically-relevant ways. To achieve these goals, we will also develop new methods to enable the repeated measurement of diverse neuronal activities that change dramatically over the course of human brain development. Finally, we will leverage our insights to study pathogenic mechanisms in a disease model of severe developmental delay and epilepsy in which neurotransmission is impaired. The tools and approaches established here may be readily adapted to cell culture models of other organ systems for which neuronal inputs are important. If successful, this New Innovator proposal will lead to ground breaking discoveries about activity-dependent maturation and will leverage those insights to unlock more advanced stages of human brain development to enable a new avenue of preclinical research to interrogate neurological disease.

PROJECT NARRATIVE Tri-dimensional (3D) cellular structures derived from human stem cells have been shown to recapitulate many aspects of fetal brain development, however, efforts to use these systems to study later stages of development have been frustrating. In order to improve the maturity and relevance of this model system for the study of more advanced brain functions, we will implement several methods systematically and in parallel to stimulate and measure activity in the cultures over extended periods of time. This intervention is expected to lead to fundamental insights into the role of activity in brain development and to enable a new avenue of preclinical research that leverages human models for the interrogation of neurological disease.