“Axonal Length Determines distinct homeostatic phenotypes in human iPSC derived motor neurons on a bioengineered platform”

Abstract

Stem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). The axons of these neurons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. It follows that shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here we directly address this issue by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, permitting systematic investigation of the effects of length on human axonal biology for the first time. With this approach, we reveal a link between length and metabolism in human MNs in vitro, where axons above a threshold size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation and mitochondrial homeostasis. Our findings specifically indicate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs in order to sustain long axons. Our findings have critical implications for in vitro modelling of several neurodegenerative disorders and reinforce the importance of modelling cell shape and biophysical constraints with fidelity and precision in vitro.

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

“…The well dimensions were homogenous and faithful to CAD specifications throughout the print sizes down to 50 µm in Y (Figure S6) . These PDMS stencil-well devices were placed on the extra cellular matrix (ECM) coated and dried micropatterned surface with axonal guidance grooves 46 , and the iPSC-derived MN cell suspension was manually pipetted into the dry wells of the device. To avoid potential air pockets in the smaller wells, as it is common for non- functionalised PDMS, we performed oxygen plasma treatment on stencils prior to cell “dry plating” ( Figure S7).…”

“…We decided to use these types of devices, using human induced pluripotent stem cell-derived motor neurons (MNs) as a cell model system, for an initial proof-of-principle of our optimised SOLID protocol, based on an engineered platform we recently developed for MN cultures using a micropatterned substrate to facilitate axonal elongation 35 .…”

“…We decided to use this application, using human induced pluripotent stem cell derived motor neurons (MNs) as a model system, for an initial proof-of-principle of our optimised SOLID protocol, based on an engineered platform we recently developed for MN cultures using a micropatterned substrate to facilitate axonal elongation 46 . We combined and optimised this platform with our SOLID moulding protocol to create a tailored plating strategy for investigating hiPSC-derived MN behaviour with control over cell location and orientation.…”

“…Despite these limitations, we tested if the topography is sufficient to align axonal elongation. We plated MN neural aggregates 39 in a SOLID-manufactured stencil device on SOLID-manufactured grooves following the 'dry plating' protocol (Figure 4B). Visualisation of axons (β-III-Tubulin) after 7 days of culture revealed an alignment with the topography throughout the axonal length (Figure 4C-D).…”