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.

Local actuation of organoids by magnetic nanoparticles

“…We also demonstrate local patterning enhancement in neural tube and paraxial mesoderm organoids, suggesting a broader role of actuation in enhancing patterning. While no preferential direction was previously observed in neural tissue under exogenous actuation 14,35 , our results demonstrates that local actuation guides directional patterning bias. Moreover, while we demonstrate the use of static magnets for actuation, it is conceivable to stimulate the magnetoids through an large number of magnetic configurations stemming from static or electro-magnets, providing design freedom for the user to fit the needs of the specific application.…”

“…A 100 μL 2 kPa Polyethylene glycol (PEG) hydrogel was prepared as previously described 14 for each condition. The 10 μL cell suspension 14 was replaced by a 10 μL aggregate suspension. 10 μL of aggregate-containing hydrogels were placed in wells of a 96-well-plate.…”

“…We found that increasing mhPSC proportion increased MagC volume and by extension the resultant force ( Fig.1d ). To ensure the embedded MagCs do not disrupt the magnetoid cytoarchitecture, an important consideration for correct differentiation and growth 13,14 , we investigated the cytoskeleton arrangement in day 11 hNTmOs with varying mhPSC proportions ( Fig.1e-f ). On average, low MagC volumes of ∼5.7×10 3 μm 3 correlated with single lumen magnetoids indicating correct apicobasal polarization ( supplementary Fig.4 ).…”

“…Mechanical stresses have been previously shown to guide cell fate specification and patterning in mouse 13,32 and human 14 NTOs. We therefore thought to investigate domain specification in our hNTmOs to evaluate mechanically-driven bias in patterning.…”

“…To investigate the forces generated by the MagCs, we generated human neural tube magnetoids (hNTmOs) following a previously described human neural tube differentiation protocol 14 . We first evaluated the magnetic field produced by a static magnet placed adjacent to a standard well-plate.…”