Assistant Professor Massachusetts Institute of Technology, United States
Introduction:: Peripheral nerve injuries are among the most prevalent injuries, seen in approximately 2.8% of trauma patients[1]. In prior literature, scientists and engineers have modeled the peripheral nerve and its associated musculature with the intent to understand the mechanism behind nerve regeneration better. Despite the large amount of data surrounding neuromuscular systems it is still not well understood how the balance between biochemical and mechanical stimuli aids in neurite growth and innervation. Recent research by Rousseau et al. (2023, in press), demonstrated that optically exercising a genetically modified tissue engineered Channelrhodopsin-2(ChR2) volumetric muscle graft on a rat model led to an increase in neurotrophic factors through proteomic analysis[2]. Moreover, histological evidence showed that the implanted tissue engineered muscle had been innervated by native axons. The proteomic analysis shows an upregulation of growth factors compared to the non-exercised tissue engineered graft. Our study decoupled the biochemical and mechanical effects in an in vitro study to further study neurite behavior.
Materials and Methods:: The base platform that we used to image and experiment with was a fibrin hydrogel-seeded glass-bottom well plate. Our co-culture consisted of embryonic stem cells from a genetically modified mouse, HB9::GFP, differentiated into motor neuron spheroids and C2C12 murine myoblasts with a ChR2-tdTomato modification, shown in Figure 1. To isolate the biochemical effects of exercised muscle we seeded the differentiated spheroids on day 8 of neural differentiation onto the fibrin hydrogel and added exercised muscle media. The exercised muscle media was made by optically exercising a differentiated monolayer of ChR2 C2C12s in complete neural differentiation media for 30 minutes at 1Hz and then centrifuged to remove any floating myoblasts. The neuronal culture was imaged every day for eight days focusing on the GFP signal from motor neurons. The same experiment was also performed with a neuromuscular co-culture to asses the combined effort of the biochemical and mechanical impact of muscle exercise on neurite growth. In the future, we plan on applying our magnetic stimulation protocol on the neuron monoculture to isolate the mechanical impact.
The HBG3 embryonic stem cells were differentiated with a base formulation of 50%(v/v) Neurobasal and 50%(v/v) Advanced DMEM/F12, supplemented with 10%(v/v) KnockOut Serum Replacement, 1%(5000U/mL) penicillin-streptomycin, 1%(v/v) L-glutamine, and 0.1mM β-mercaptoethanol. The neural differentiation media was supplemented with 1µM of purmorphamine and retinoic acid. In the final days of differentiation, 10ng/mL of glial-derived neurotrophic factor and ciliary neurotrophic factor were added.
Results, Conclusions, and Discussions:: The preliminary results show a substantial increase in neurite outgrowth when comparing the control and supplemented media group as shown in Figure 2. The individual curves in Figures 2B and 2E show not only an increase in max neurite growth but also in the migration area of neurites. The representative images also show a denser network of neurites surrounding the embryoid body.
We have ongoing co-culture experiments analyzing the growth patterns of neurites within a 2D musculature. As Figure 3 shows, there is a preferred directional alignment for the axons to grow, more specifically along the local alignment of the differentiated myofibrils. In the future, we plan on applying a magnetically actuated hydrogel platform we have developed (Rios* & Bu*, Manuscript in submission) on the neuron monoculture to isolate the mechanical impact (Figure. 1E).
For future works, we plan on performing glutamate stimulation and alpha-bungarotoxin staining on the neuromuscular junctions to quantify the functional differences between the experimental conditions. Moreover, utilizing RNAseq to look at how the global transcriptome is affected by exercise, biochemical stimulation, and mechanical stimulation separately and together. Using this model we will investigate how exercise impacts neuromuscular junction formation, maturation, and response to injury.
