Actuation Enhances Patterning in Human Neural Tube Organoids

Abstract

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

Organoids as a new model system to study neural tube defects

“…However, other factors may also need to be considered. For example, a recent study has shown that mechanical force of culture environment can also affect neural tube organoids growth and patterning 73 . All these three improvements will make neural tube organoids more reliable and more similar to the in vivo neural tube.…”

Neuroepithelial organoid patterning is mediated by Wnt-driven Turing mechanism

“…Organoids have often been used as simplified models of patterning and have allowed insight into the underlying biochemical [41][42][43][44] and mechanical 1,20,45,46 factors that control cell fate specification and morphogenesis. We and others have shown that NTOs display patterned FPs 9,11,12,20 which emerge, remarkably, despite the lack of the notochord, which in vivo acts as a spatial reference frame for FP patterning 28,47,48 through the secretion of SHH morphogens. This suggests that in vitro epithelial patterning follows mechanisms that might differ from those employed in vivo.…”

“…In order to identify the inhibitor species in the hNTO system, we performed receptor-ligand interaction analysis 31 on an scRNAseq dataset obtained from dissociated hNTOs at day 5, corresponding to hNTOs after RA-SAG exposure but before FP induction, and at day 11, after FP induction and patterning 20 .…”

Development in a Dish—In Vitro Models of Mammalian Embryonic Development

“…Anteroposterior and dorsoventral identity could be modulated by the activation of retinoic acid or sonic hedgehog signaling ( Ranga et al, 2016 ). In addition, morphogenetic changes inducing neural tube closure were mimicked in vitro , which improved neural differentiation and patterning of NTOs derived from hPSCs ( Fattah et al, 2020 ; Figure 2 ).…”