Comparing Transcriptome Profiles of Neurons Interfacing Adjacent Cells and Nanopatterned Substrates Reveals Fundamental Neuronal Interactions

Abstract

Developing neuronal axons are directed by chemical and physical signals toward a myriad of target cells. According to current dogma, the resulting network architecture is critically shaped by electrical interconnections, the synapses; however, key mechanisms translating neuronal interactions into neuronal growth behavior during network formation are still unresolved. To elucidate these mechanisms, we examined neurons interfacing nanopatterned substrates and compared them to natural interneuron interactions. We grew similar neuronal populations under three connectivity conditions, (1) the neurons are isolated, (2) the neurons are interconnected, and (3) the neurons are connected only to artificial substrates, then quantitatively compared both the cell morphologies and the transcriptome-expression profiles. Our analysis shows that whereas axon-guidance signaling pathways in isolated neurons are predominant, in isolated neurons interfacing nanotopography, these pathways are downregulated, similar to the interconnected neurons. Moreover, in nanotopography, interfacing neuron genes related to synaptogenesis and synaptic regulation are highly expressed, that is, again resembling the behavior of interconnected neurons. These molecular findings demonstrate that interactions with nanotopographies, although not leading to electrical coupling, play a comparable functional role in two major routes, neuronal guidance and network formation, with high relevance to the design of regenerative interfaces.

Self-Assembling Hydrogel Structures for Neural Tissue Repair

“… 93 , 145 , 164 − 166 For example, dorsal root ganglia cells increase the maximum length of their neurites by 82% when exposed to core–sheath nanofibers. 167 Baranes et al 168 showed that nanotopographies altered gene expression profiles of primary neurons isolated from medicinal leaches, upregulating axon-guidance signaling pathways, synaptogenesis and synaptic regulation, resembling the behavior of interconnected neurons. Human embryonic stem cells (hESCs) can be differentiated into a neuronal lineage by exposing them to an aligned ridge pattern, without the need for other differentiation-inducing agents.…”

Manipulation of Axonal Outgrowth via Exogenous Low Forces

“…Nanopatterned scaffolds (NSs) are a common way to study the mechanical effects of substrates on cells. They are used to mimic the extracellular environment in order to evaluate the response of neurons to substrate stiffness [141], to study the role of mechanical tension in determining the final morphology of neuronal networks [96], or to study the process of neural guidance on non-flat substrates [142].…”

“…Stiffness in the NGC can be used as a mechanical signal modulating cell behavior [141,153]. Mechanical inputs deriving from the stiffness of the fibers in the NGC can be delivered by modifying the concentrations and blending of polymers [154] and the porosity [155], dimensions [156,157], crystallinity [157] and anisotropy [142] of an individual fiber. Nanofibers can be used inside the NGCs to allow axons to grow through these fibers as a guide for tissue regeneration [158].…”

Neuro‐taxis: Neuronal movement in gradients of chemical and physical environments

“…The interactions of neurons with topographical cues, eliciting morphological changes, resulted in the morphological similarity to the neurons contacted with other neurons (Baranes, Chejanovsky, Alon, Sharoni, & Shefi, 2012; Baranes, Kollamar, Chejanovsky, Sharoni, & Shefi, 2012) (Figure 4c). The intracellular cytoskeletal dynamics were investigated in detail by transcriptome‐expression profiling (Baranes et al., 2019). Using both the functional annotation analysis for the top‐50 differentially expressed genes and the pairwise enrichment analysis between the culture conditions (N: non‐contact; C: contact; L: lines), the upregulated or downregulated genes were compared (Figure 4d).…”