Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34)

Abstract

Introduction: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy.

Background: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration.

Methods: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons?

Conclusion: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

Keywords: extracellular vesicles; motor neuron; muscle; neurite outgrowth.

Fig. 1

Time course of myosin expression in serum-free AIM-V media (Invitrogen). After propagating cells in DMEM with foetal bovine serum the cells are switched to Aim-V. Panel (A) shows staining for myosin in the early differentiation phase, showing the beginning of myotube formation. Panel (B) shows the expression of myosin begins by day 3 and is still present by day 7.

Fig. 2

Characterization of C2C12 extracellular vesicles. (A) The Nanosight instrument and nanoparticle tracking analysis revealed vesicles primarily 100 nm and less with a peak around 54 nm. (B) Western blot showing the presence of the tetraspanin CD-63 which is a standard marker for extracellular vesicles (24 µg of total protein loaded per lane). (C) Western blot showing significant reduction of the endoplasmic reticulum marker calnexin in the extracellular vesicle fraction compared to the cell lysate which validates the enrichment procedure that was used to isolate the extracellular vesicles (12 µg of total protein loaded per lane). See Supplementary file for uncropped images of the Western blots.

Fig. 3

The motor neuron cell line NSC-34 was exposed to fluorescently labelled vesicles for 3 hours, then fixed and stained for neurofilament to label neurite processes. There was extensive uptake of the labelled vesicles by the neurite processes with some indication of accumulation around perinuclear regions. Size bar=50 microns.

Fig. 4

NeuriteQuant analyses of motor neuron process outgrowth. (A and B) Raw images from control (no vesicles) and vesicle-treated cultures. (C and D) Processed image after running NeuriteQuant program. (E and F) Higher power image of inset boxes from C&D to show various aspects of the quantified data. Motor neuron cell bodies are shown in yellow, neurite processes are shown in blue, with their attachment points to the cell bodies indicated by small white circles, and their end points indicated by small red circles. Size bar in A=100 microns and in E=30 microns.

Fig. 5

NeuriteQuant analysis (mean±SEM). Motor neuron cells were cultured for 4 days in the presence or absence of varying concentrations of vesicles purified from conditioned media of muscle cells as described in the text. Semi-automated analysis of average neurite length per neuron (A), and the complexity of the neurites as judged by the number of branches per neurite (B) indicates a significant increase in length and complexity for all vesicle-treated groups compared to control (F=0.04, t-tests <0.05). (C) There was a dose-response effect on cell body size due to vesicle treatment compared to control that showed a trend at 100 ng/µl (p=0.06) and reached statistical significance with the 200 ng/µl vesicle group (F=0.02, t-tests =0.06 and < 0.01, respectively). (D) The total cell count also displayed a dose-response effect that reached significance with the 200 ng/µl vesicle group (F=0.05, t-test=0.03). The graphed results for each outcome measure are from conditioned media from 2 independent C2C12 myotube cultures (i.e. 2 biological replicates), with 2 independently prepared batches of extracellular vesicles from each media preparation (i.e. biological replicates), with 8 repetitions of each concentration (i.e. technical replicates). In total, the data represent NeuriteQuant analysis for >6,000 motor neurons. The “N” used for statistics was 4, obtained by the number of biological replicates of vesicle preparations.

Fig. 6

CellTiter-Glo viability assay of motor neurons after 4 days in culture. The control treatment refers to cells grown in media alone, without the addition of vesicles. There was a strong dose-response effect on cell survival compared to control with a trend at the lowest concentration (11.5 ng/µl), and then significantly greater effects with each increased concentration of vesicles (F<0.0001; t-tests as indicated on the figure; mean±SEM). The graphed results are from conditioned media from 3 independent C2C12 myotube cultures (i.e. 3 biological replicates), with 2 independently prepared batches of extracellular vesicles from each media preparation (i.e. biological replicates), with 12 repetitions of each concentration (i.e. technical replicates). The “N” used for statistics was 6, obtained by the number of biological replicates of vesicle preparations.