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

Abstract

Biophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment. However, the complexity and experimental challenges capturing such early stages of development, and ethical concerns associated with human embryo and fetal research, limit our understanding of how these factors affect human brain organogenesis. Human cerebral organoids (hCO) are attractive models due to their ability to model important brain regions and transcriptomics of early in vivo brain development. Furthermore, they provide three-dimensional environments that better mimic the in vivo environment. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Establishing links between spatial factors and hCO development will require the development of new approaches. Here, we investigated the effects of early geometric confinements on transcriptomic changes during hCO differentiation. Using a custom and tunable agarose microwell platform we generated embryoid bodies (EB) of diverse shapes and then further differentiated those EBs to whole brain hCOs. Our results showed that the microwells did not have negative gross impacts on the ability of the hCOs to differentiate generally towards neural fates, and there were clear shape dependent effects on neural lineage specification. In particular, we observed that non-spherical shapes showed signs of altered neurodevelopmental kinetics and favored the development of medial ganglionic eminence-associated brain regions and cell types over cortical regions.The findings presented here suggest a role for spatial factors in brain region specification during hCO development. Understanding these spatial patterning factors will not only improve understanding of in vivo development and differentiation, but also provide important handles with which to advance and improve control over human model systems for in vitro applications.

Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids

“…Physical forces, such as shear stress, gravity, and cyclic stretch affect mechanosensitive pathways or change the expression levels 22 . Recently, the transcriptomic analyses of cerebral organoids showed that mechanotransduction-associated genes including integrins, β-catenin, Wnt, and Delta-like pathway genes change under physical stress conditions 98 . Thus, organoids exposed to varying magnitudes of hydrodynamic forces are expected to display differences in structural and functional features during development.…”

SOL3D: Soft-lithography on 3D vat polymerised moulds for fast, versatile, and accessible high-resolution fabrication of customised multiscale cell culture devices with complex designs

“…Next, we tested whether the ease of available prototyping using our optimised SOLID protocol could enable investigation and manipulation of the fundamental behaviour of complex iPSC-derived MN cultures. It has been shown that aggregation of cells using different geometries has an influence on the signalling environment and patterning of aggregates 38 . We therefore designed and fabricated moulds for PDMS stencils with 3 different well shapes: rectangular, circular, and triangular, to create geometrically constrained neural aggregates.…”