Optimization of cerebral organoid differentiation from different lines of human pluripotent stem cells

The cerebral cortex is a specialized region of the brain responsible for higher neurological functions such as sensory processing, learning and memory, cognition, and abstract thinking. This region is disproportionally enlarged in humans and other primates, and thought to be one of the factors that allows for our enhanced intellectual capacities compared to lower species such as rodents. Hence, rodent models are not ideal tools for studying the primate-specific features that drive brain growth and complexity. Several studies have shown that human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) have the ability to form three-dimensional cortex-like structures called organoids that recapitulate distinct aspects of human brain development. However, our and others' results indicate that cortical organoid formation is variable and often not reproducible, differing between hESC and hIPSC lines and maintenance methods. One of the biggest influences that we have found is growth under mouse embryo fibroblast (MEF)-dependent vs. independent conditions. Maintenance with MEFs permitted robust and reproducible organoid differentiation from either hESC or hIPSC. Importantly, cells initially grown under feeder-free conditions can be entrained to produce organoids when passaged with MEFs. Moreover, the positive effects of MEFs on organoid differentiation require sustained MEF-dependent culture. Organoids differentiations have been checked by immunostaining for cortex main markers. The transcriptome of cells cultured under different conditions, analyzed by RNA-seq to identify hallmarks of successful cortical organoid differentiation. A better understanding of how these different maintenance conditions influence organoid development will help predict experimental outcomes and improve the application of these methods for modeling normal and diseased human brain development.

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