Improving Brain Organoid Models by Mediating Metabolic Dysregulation

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Project Summary There is currently an unmet need for accurate model systems of the human brain to study its cellular and molecular features. The cerebral cortex regulates our cognitive capacity, yet the cellular diversity, circuit formation, and function that establish this potential, largely remains a mystery. The cortex is expanded in humans compared to other species; it contains more cellular diversity and abundance, making model organisms limited for translational studies. Brain organoids provide access to human cells for experimental manipulation and recent studies have suggested that organoid models may act as a proxy for the human brain when studying developmental trajectories, circuitry, and a variety of complex neurological diseases. However, using single cell RNA sequencing we performed validation studies of cortical organoid models compared to primary human cortex cells and we discovered several significant deficiencies in organoid cells. First, organoids do not make the same refined cell types with clear gene expression programs observed during normal cortex development. They also lack molecular maturation networks observed endogenously. As organoid cells do not make the specific, refined cell types we observe in the human brain, these models are severely limited when aiming to model circuit activity and disease. Circuits are comprised of highly specialized neuronal subtypes that target one another to wire defined connections and ultimately produce appropriate physiological activity. We require organoid models that make specific cell types that can mature enough to form the building blocks of canonical circuits to enable the study of neural connectivity moving forward. In addition to cell type deficiencies, all organoids assayed, regardless of derivation method, express high levels of metabolic stress. Using transplantation studies, we identified that cellular stress is linked to the in vitro environment, it negatively impacts cell type identity, and it can be reversed by removal from culture conditions. My proposed studies will directly address the current technical limitations of organoid models by intervening in the metabolic dysregulation with the goal of improving cell type specification and maturation. First, I will target glycolytic and endoplasmic reticulum stress pathways, using small molecules to inhibit these processes and by modulating glucose and oxygen concentrations. I will also utilize knowledge of relevant signaling pathways in human cortex development, such as the LIF signaling pathway, which is an important regulator of a human-enriched population of cortical stem cells. Second, I will work to identify the relevant non-neural cells required for normal metabolic regulation endogenously, which are absent from organoid models. To work toward this aim I will use transplantation of human organoid-derived cells into the mouse cortex environment and transplant relevant vascular and immune cells into cortical organoids in vitro. Together, these studies will improve current technical limitations in metabolism and cell type impairment to ensure that organoids more accurately reflect the cellular diversity of the human cortex and provide a tractable model system for the study of human neuroscience.