Human cerebral organoids (hCO) are in vitro models of the brain derived from pluripotent stem cells. hCOs are attractive for studying human brain development as they capture some complex structures of the brain and bypass ethical concerns of human in vivo studies. hCOs as a model system are being continually improved, with interest in factors that affect their development. Prior studies show that alteration of mechanical forces and spatial patterning of stem cells biases their differentiation. In order to understand the effects of mechanical cues and spatial factors on hCO development, we investigated the impact of early geometric confinement on whole transcriptomic changes during hCO differentiation. Embryoid bodies, generated at the beginning of hCO protocols, were forced into geometric confinement using four microwell shapes: round, C, peanut, and butterfly. We hypothesized that shape would influence downstream hCO development and brain region specification, which would be reflected in differential RNAseq patterns. Culturing hCOs in 96-wells were compared to soft-lithographically micropatterned agarose microwells to account for potential effects of changing material properties of the growth environment. The microwell hCOs met the development milestones that occur in the 96-well hCOs. We then investigated the effect of microwell geometries on hCO development. In comparison to the round microwells, all other microwells exhibited transcriptomic differences. The hCOs grown in butterfly microwells had the highest number of differentially expressed genes, followed by the peanut and then the C microwell hCOs, and all favored the development of ventral brain regions and cell types over dorsal cortical regions. Interestingly, the amount of differentially expressed genes correlated with the number of stress points applied to the EBs in each shape. The study suggests that spatial factors and geometric confinement of the EB growth environment play a role in the brain region specification of hCOs and could have physiological relevance in our understanding of how nascent embryonic features affect fetal brain development. This approach will provide methods both to study mechanobiological regulation in neurodevelopment and to control hCO differentiation.

Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids

ACS Spring 2022

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