Nanowired Neurospheres for Improved Electrophysiological Maturation

Introduction: Compared to conventional two-dimensional cultures, three-dimensional spinal motor neurospheres exhibit improved physiological relevance as model systems – a pertinent example lies in neuromuscular junction organoids that replicate pathologies of amyotrophic lateral sclerosis. Moreover, stem-cell spheroids demonstrate improved translational potential for pathologes like traumatic spinal cord injury. However, these neurosphere cultures are currently limited by lengthy timelines of functional maturation, and do not completely replicate in vitro spinal neuron circuits. Previous research has demonstrated the ability for a network of silicon nanowires to improve the bioelectrical synchronization of cardiac spheroids, both in vitro and post-transplantation in vivo. Here, we demonstrate the stable integration of silicon nanowires—a conductive, biodegradable, high-aspect-ratio nanomaterial—within neurospheres with the goal of improving their electrophysiological maturation. Within these constructs, nanowire-neuron interfaces could bioelectrically couple adjacent, immature motor neurons. Nanowired neurospheres are thus hypothesized to exhibit more synchronized electrophysiological activity and improved synaptogenesis in the early stages of neuronal maturation – eventually, such effects could lead to the formation of physiologically-relevant spinal motor neuron circuits in vitro.

Materials and

Methods: Cervico-thoracic motor neurons were generated from H9 human embryonic stem cells through a staged, modular differentiation process. Briefly, human embryonic stem cells underwent Hox patterning for 120 hours along the rostrocaudal axis, followed by dorsoventral patterning towards motor neuron fates. Preliminary spinal neuron induction was accomplished via 7 days of Notch inhibition. Degenerately doped, p-type silicon nanowires were fabricated through gold-nanoparticle-catalyzed vapor-liquid-solid growth in a chemical vapor deposition chamber, utilizing silane gas (SiH4) as the silicon source, hydrogen as the carrier gas, and diborane (B2H6) as a boron dopant source. Reaction conditions were held constant at 20 Torr, 440 °C, and a feed doping ratio of 500: 1 (SiH4: B2H6 molecules). We particularly optimized 60- and 120-minute vapor-liquid-solid growth periods to minimize nanowire overcoating. We also coated a sample of 60-minute silicon nanowires with laminin by incubating the sample in a 0.5 µg/mL solution at room temperature for 1 hour. After synthesis and treatment, silicon nanowires were harvested via 15 seconds of 37 kHz sonication at 50% power. Motor neurons were singularized, combined with the harvested silicon nanowires at a 1:1 ratio, and seeded into agarose-cast microwells to facilitate neurosphere self-assembly at a density of 1500 cells/neurosphere (Figure 1A). The density of seeded silicon nanowires was quantified post-hoc via darkfield microscopy on a hemocytometer. Neurosphere viability was assessed via brightfield microscopy and viability staining. Nanowire localization and functional maturation were assessed via immunocytochemical staining, laser-scanning confocal microscopy, and transmission electron microscopy of resin-embedded neurosphere sections.

Results, Conclusions, and Discussions: Our experiments focused primarily on motor neurospheres integrated with silicon nanowires with different lengths (60-minute, 120-minute nanowires) and coatings (laminin-coated, uncoated nanowires) up to 21 days in vitro. We found no significant effects of nanowire introduction, under any nanowire condition, on the extended viability of nanowired neurospheres. Laser-scanning confocal microscopy revealed extensive nanowire integration throughout the neurospheres, with significant co-localization between nanowires and axon projections (Figure 1B). In addition, transmission electron microscopy of nanowired neurosphere sections showed intracellular infiltration of nanowires within motor neurons (Figure 1C).
After further image analysis, we concluded that a laminin coating did not significantly affect nanowire localization or ejection within the X-Y plane of the neurospheres. However, we observed a statistically-insignificant increase in total nanowire retention in neurospheres integrated with laminin-coated nanowires. When assessing synaptogenesis, we identified colocalization of synapsin puncta with regions of concentrated nanowires. Across the entire neurosphere, we also quantified a statistically-insignificant increase in the total number of synapsin puncta in nanowired neurospheres compared to controls. We posit that electrical coupling of motor neurons through this electrically-conductive nanowire network directly leads to increased synaptogenesis.

We have thus demonstrated successful integration of p-type, degenerately-doped silicon nanowires within 3D motor neurospheres. From confocal and transmission electron microscopy, we have identified that nanowires infiltrate intracellularly and form electrical bridges between distinct axon fibers. We hypothesize that these nanowire bridges mimic electrical synapses, which play a crucial role in the postnatal maturation of motor circuits in vivo. Therefore, over extended culture periods, we anticipate that nanowired neurospheres could exhibit accelerated formation of oscillatory, circuit-like neuronal activity. The electrical conductivity of the nanowire network will also augment the effect of external electrical stimulation on these 3D culture systems to facilitate further neuronal maturation.

Moreover, our method of forming nanowired neurospheres can be extended to organoid models or neuronal stem-cell spheroids without significant alterations. Thus, this preliminary data sets the foundation for future studies towards characterizing the function of electrical synapses in motor circuits, improving models for spinal neuron pathologies (e.g., amyotrophic lateral sclerosis), and enhancing cell-transplantation therapies for traumatic injuries (e.g., electrophysiological training of spinal cord grafts).

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