Author(s): Emmy Li, University of California, San Francisco; Emmy Li, University of California, San Francisco; Mark Koontz, University of California, San Francisco; Nina Draeger, University of California, San Francisco; Steven Boggess, University of California, San Francisco; Erik Ullian, University of California, San Francisco; Martin Kampmann, University of California, San Francisco //

ABSTRACT: The interactions between glial and neuronal cells play a large role in disease. Both astrocytes and microglia, for example, can become reactive in neurodegenerative diseases, leading to a response that may be detrimental to neuronal survival. Though many studies have investigated how certain glial cell types may independently contribute to disease, how interactions between neurons and glia may make neurons more vulnerable is less well understood.    Thus, we generated 3D iPSC-derived neuron-astrocyte-microglia assembloids (iAssembloids) to model how neuronal and glial cells interact. Making use of transcription factor-based neuronal and microglia differentiation protocols, iAssembloids are reproducible and easy to generate in a high-throughput manner, making them suitable for large-scale screening applications. In addition, transcriptional profiling and functional studies have shown that iPSC-derived neurons cultured in iAssembloids have higher expression of axon guidance-related genes and are more electrophysiologically active than standard monoculture iPSC-derived Ngn2 neurons. Therefore, iAssembloids can provide a more physiologically relevant handle on probing glia-neuron interactions.     With these iAssembloids, we performed pooled CRISPRi-based functional genomics screens in the iPSC-derived neurons and identified pathways that can promote or hinder neuronal survival specifically in the iAssembloid context, but not in monoculture. Pathways that we have identified include those important for neuronal activity and metabolism. By using CRISPRi-based screens in combination with single-cell RNA sequencing, we can also investigate transcriptomic changes that occur in the perturbed iPSC-derived neurons in the iAssembloids compared to monoculture. Using both these tools allows us to further understand the specific mechanisms by which these pathways are differentially affected in monoculture versus iAssembloids.     We aim to expand CRISPRi-based screens in iAssembloids to compare neuronal phenotypes when co-cultured in different isogenic, disease-related glial environments (eg. APOE3 vs APOE4). By performing CRISPRi-based screens in assembloids derived from patient iPSCs with genetic risk variants, we can start to address questions about diseases in which glial cells are hypothesized to play a large part as well as provide a new method to test possible therapeutics.

Source of Funding: NDSEG Fellowship