An Engineered Hydrogel Platform to Improve Neural Organoid Reproducibility for a Multi-Organoid Disease Model of 22q11.2 Deletion Syndrome

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Project Summary l The complexity of the human brain and lack of adequate models severely hinders our ability to understand mechanisms guiding neurodevelopment and neurodevelopmental disorders (NDDs), necessitating an innovative bioengineered approach for improving in vitro organotypic models of the human brain. Recent advances in stem cell-based neural organoids enable the formation of assembloids, which are fusions of organoids representing different brain regions. However, current approaches remain limited in the ability to properly recapitulate native brain cytoarchitecture and maturation within these organoids and also result in large heterogeneity due to the lack of a well-defined matrix. The proposed research seeks to resolve these critical issues by creating a reliable and reproducible in vitro environment for neural organoid culture to study aspects of neurodevelopment and NDDs that have been difficult to achieve with current platforms. To do this, I will 1) assess the effect of matrix biochemical cues for improving neural organoid architecture and maturation; 2) define the role of matrix stress relaxation and confinement on organoid growth; and 3) leverage the engineered hydrogel platform to study impaired interneuron migration in a disease model of 22q11.2 deletion syndrome (22q11DS). I hypothesize that the experiments described in my proposal will show that matrix-derived biochemical and biophysical signaling will allow for more robust neural organoid culture that better recapitulates the architecture and maturation of the human brain compared to conventional neural organoid models. I also hypothesize that fusion of 22q11DS neural organoids within engineered hydrogels will robustly demonstrate dysregulated interneuron migration mediated by the deletion of DCGR8. Of note, interneuron migration is a phenomenon that does not occur in murine systems and therefore cannot be studied using conventional murine models. To test this hypothesis, I will utilize a minimal matrix (HELP) to culture brain region-specific neural organoids derived from induced pluripotent stem cells (iPSCs) from healthy and 22q11DS patients. I will perform robust characterization of neural organoid architecture, maturation, and growth rate in response to tuning matrix biochemical and biophysical properties. I will also assess the ability of interneurons from 22q11DS patients to migrate into the dorsal forebrain by establishing a dorsal–ventral forebrain assembloid disease model. Together, these results will be critical for engineering a platform that is both permissive and instructive for robust and efficient neural organoid culture. In addition to expanding my scientific technical skills, my training plan includes development of mentorship, scientific writing, and presentation skills; training in research ethics; and enhancement of collaboration skills through a series of on-campus courses, workshops, and seminars as well as off-campus conferences. Altogether, this research proposal will empower me to become an independent, productive research scientist as I leverage the self- organizing capacity of stem cells and the tunable capacity of engineered materials to develop more human- relevant neural disease models.