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

Abstract

Background Human cerebral organoids (hCO) are attractive systems due to their ability to model important brain regions and transcriptomics of early in vivo brain development. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Interestingly, one of the main advantages of hCOs are that they provide three dimensionality that better mimics the in vivo environment; yet, despite this central feature it remains unclear how spatial and mechanical properties regulate hCO and neurodevelopment. While biophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment, much of this work investigated two dimensional systems or relied on correlative observations of native developing tissues in three dimensions. Using hCOs to establish links between spatial factors and neurodevelopment will require the use of new approaches and could reveal fundamental principles of brain organogenesis as well as improve hCOs as an experimental model. Results 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 mimicking several structures from embryogenesis and neurodevelopment 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 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. Transcriptomic analysis suggests these mechanotransducive effects may be mediated by integrin and Wnt signaling. Conclusions 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.…”

Spatial confinement toward creating artificial living systems

“…Sen et al reported that early geometric confinement could cause transcriptomic changes in human cerebral organoids differentiation. 158 Ruiter et al encapsulated iPSCs-derived kidney organoids in hydrogels with controllable stiffness or stress relaxation time. 159 It was found that organoids cultured in softer hydrogels were more mature compared to those cultured in high-stiffness hydrogels.…”

Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designs

“…Next, we tested whether the ease of available prototyping using our optimised SOL3D 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 [ 45 ]. We therefore designed and fabricated moulds for PDMS stencils with 3 different well shapes: rectangular, circular, and triangular, to create geometrically constrained neural aggregates.…”