Members of SCTL presented a poster at ISSCR 2020 Virtual. The poster is entitled: Improved Cell Survival and Cytoprotection Enhance Reproducibility and Proper Differentiation of Cerebral Organoids Derived from Human Pluripotent Stem Cells.
Authors (underlined, presenting): Seungmi Ryu, Claire Malley, Pei-Hsuan Chu, Ben Ernest, Carlos Tristan, Vukasin Jovanovic, Tao Deng, Anton Simeonov, Ilyas Singeç
More information about ISSCR 2020 Virtual.
Self-organizing organoid models from human pluripotent stem cells (hPSCs) recapitulate some aspects of brain development and function. However, current protocols are hampered by uncontrolled cell death, considerable organoid-to-organoid heterogeneity, and lack of standardization. We found that ROCK inhibitor Y-27632, the most widely used reagent to improve cell survival during embryoid body (EB) formation, is deficient in preventing cellular stress and apoptosis, leading to nonoptimal organoid formation. Here, we used a newly developed small molecule cocktail termed “CEPT” (Chroman 1, Emricasan, Polyamines, Trans-ISRIB according to Chen et al., 2019, bioRxiv), which greatly enhanced cell survival and cell quality during EB formation. The data demonstrated that improved cell survival during the early stages of EB formation has long-lasting consequences affecting not only total cell numbers and organoid size but also enhanced neuronal differentiation of organoids cultured up to 60 days. Single-cell RNA sequencing from two-month old organoids revealed that CEPT-generated organoids vs. Y-27632 treatment contained higher expression levels of neuronal markers representative of different cortical layers. Moreover, transcriptomic analysis of CEPT-generated organoids indicated significantly higher correlation to datasets from the developing human brain. Importantly, CEPT-generated organoids resulted in experimental reproducibility based on RNA sequencing analysis across individual organoids. In summary, our study identified uncontrolled cell death at the onset of organoid formation as a critical quality control determinant and demonstrated that using the newly developed CEPT cocktail dramatically improved morphogenesis, neuronal differentiation, and overall reproducibility of cerebral organoids.