Abstract
BackgroundHuman 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.ResultsHere, 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.ConclusionsThe 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.
Highlights
Human cerebral organoids are attractive systems due to their ability to model important brain regions and transcriptomics of early in vivo brain development
Development and optimization of shape controlled Human cerebral organoids (hCO) in agarose microwells The first step in the majority of current hCO protocols is the generation of embryoid bodies (EBs) and neuroectoderm from human pluripotent stem cells [3, 24, 25]
We aimed to capture the first 5 days of EB generation after cell seeding in microwells, during which media conditions allow all germ layers to form spontaneously [6, 31]. Several design parameters such as volume and shape geometry were optimized for formation of a single EB in each microwell similar to those that were generated in U-bottom 96-well plates (Fig. 1b–d)
Summary
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. Recent progress with human cerebral organoid (hCO) systems show that these in vitro systems recapitulate many cell types and key features of the early in vivo brain making them attractive models to study human neurodevelopment [3,4,5,6,7,8,9,10,11] As a model, they are continually being improved to better mimic in vivo architectures and brain regions and to enhance reproducibility, amongst other features. While there is a relatively mature understanding of soluble factors and their effects on cerebral organoid development, the role and impacts of mechanobiological factors is unclear
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