Periodic Reporting for period 4 - MiniBrain (Cerebral Organoids: Using stem cell derived 3D cultures to understand human brain development and neurological disorders) Reporting period: 2021-07-01 to 2021-12-31 Summary of the context and overall objectives of the project The human brain is the most complex but also the most fascinating organ of all. Its 87 billion neurons arise during development following a precisely orchestrated plan. Any defect in this process can lead to profound cognitive deficiencies and severe life-long impairment. Currently, essentially all we know about human brain development is deduced from animal experiments. While it is safe to assume that the basic principles of neurogenesis are widely conserved, recent experiments have revealed a growing number of specific features that are unique to the human brain and cannot be studied in animals. The overall goal of this project is to use three-dimensional cell culture in order to elucidate how those processes are regulated and how their impairment can lead to neuro-developmental disorders. We use a tissue culture system called cerebral organoids that we have developed in 2013 and that can recapitulate brain development at a remarkable level of detail (Lancaster et al., 2013). Our goal is to recapitulate various human diseases in those organoids and to develop methodology for large-scale parallel analysis of genes that could potentially be responsible for those diseases. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first reporting period, we were already able to improve the culture system by combining it with bio-engineered scaffold material, allowing for even better reconstitution of key developmental pathways (Lancaster et al., 2017). In addition, we could reconstruct even long-range interactions between distant parts of the human brain in the organoid system (Bagley et al., 2017). This allowed us to reconstitute the long-range migration of human interneurons from the lower to the upper part of the brain. We recapitulated brain cancer, the deadliest of all brain diseases (Bian et al., 2018). We further discovered that metabolic reprograming in neural stem cells irreversibly converts them into tumor stem cells initiating malignant overgrowth (Bonnay et al., 2020). In addition, we used cerebral organoids to model the teratogenic effects of pathogenic viruses (Krenn, et al., 2021). Furthermore, we were able to ascertain that Tuberous Sclerosis, a rare neurodevelopmental genetic disorder, arises developmentally rather than only genetically (Eichmueller et al., 2022). We expect to apply our knowledge on human-specific principles in brain development and pathology to other known diseases for which no therapies exist to-date.We developed a genetic loss-of-function screening (CRISPR-LICHT) using the cerebral organoid model which allows us to screen for genes with suspected involvement in a specific human brain disorder. Not only were we able to identify microcephaly genes with this method, but we also pinpointed a specific mechanism involved in controlling the size of the brain. This mechanism affects the integrity of the tissue, and thus the brain size and was identified as one cause of microcephaly (Esk, Lindenhofer et al., 2020). A direct application of the CRISPR-LICHT is nearing completion, and a manuscript is under preparation (Chong et al., in preparation).Finally, we developed a novel cerebral organoid technology that enables the introduction of a cortical patterning axis in human brain organoids (Bosone, et al., in revision). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We are excited to report that we have achieved all aims outlined in our proposal and made groundbreaking technological innovations which led to impactful publications and acquisition of knowledge that changes our view of biology as we know it. We are building on these solid foundations by expanding the application of our methods on different diseases, pushing the boundaries of what is possible.