Notochordal Cell Conditioned Medium Inhibits Neurite Outgrowth and Maintains Neural Cell Viability
Authors: Purmessur D. , Cornejo M. C. , Cho S. , Stock I. , Iatridis J. 
Published: June 2012

Introduction

Minimally invasive therapies to treat disk degeneration focus on analgesia rather than reducing the source of pain. Painful human disks demonstrate extension of neurons that are responsive to nerve growth factor (NGF) and substance P, into the annulus fibrosus (AF) and nucleus pulposus (NP).1,2 Understanding and recapitulating the developmental processes that occur during early patterning of the spine may help to formulate symptom-modifying treatments for painful disk degeneration. The chick notochord produces chemorepulsive factors including semaphorin 3A and CS proteoglycans that inhibit axonal elongation of dorsal root ganglia.3 We suggest that notochordal cells (NCs), which play an essential role in the formation of the avascular and aneural NP, produce soluble factors that can inhibit neural extension into the disk and limit painful symptoms. We hypothesize that conditioned media (CM) from NC tissue can inhibit neuronal growth through assessment of neurite-expressing cells, neurite length, and expression of neuronal outgrowth proteins (GAP43) while maintaining viability.

Materials and Methods

Notochordal cells (NC) CM from tissue (NCT) was generated from NC-rich NP tissue from six pig spines (age 2 to 8 months), soaked separately in basal media (DMEM + 1% ITS) and conditioned for 4 days in hypoxia (1% O2, 5% CO2, 37°C). Media was filtered and the filtrate retained, resuspended in EMEM:F12 serum-free (sf) media and stored at −20°C. Human SH-SY5Y neuroblastoma cells were seeded at a density of 50,000 cells per well in a 6-well poly-d-lysine plate and incubated with EMEM:F12 + 15% FBS for 24 hours at normoxia 5% CO2 37°C. SH-SY5Y cells were then incubated for 48 hours in NCT at doses of 100 and 10% and 50% in basal media (EMEM:F12 sf media +40 ng/mL PDGF) including a basal control group. After 48 hours, images at 10× were taken per group and the percentage of neurite expressing cells and mean neurite length were calculated per field. Cell number was assessed by counting total cells per field of view, and viability was assessed using the live or dead assay. Cell images were quantified using ImageJ software. qRT-PCR was assessed using TaqMan assays for Enolase, GAP43, neurofilament, NPY, and NRP. Immunohistochemical assessment of semaphorin 3A expression was performed on porcine NC tissue from which NCT was generated (Abcam: ab45376). Statistics involved a one-way ANOVA and Tukey posthoc test with p < 0.05 considered significant.

Results

NCT significantly decreased the percentage of neurite-expressing cells at doses of 10, 50, and 100% compared to basal ( p < 0.05) (Fig. 1A). NCT also reduced mean neurite length from SH-SY5Y cells and this was significant at all doses compared to basal ( p < 0.05) (Fig. 1B). Cell viability remained high with no differences between groups (Fig. 1C). Significant increases in cell number were observed at doses of 10 and 50% (102.1 ± 5.2 and 117.1 ± 5.9) compared to basal (79.5 ± 3.1) and 50% compared to 100% (93.5 ± 5.9). No significant difference in cell number was observed at 100% compared to basal. A trend of decreased mRNA expression of neuronal outgrowth factors was observed at all doses in particular for 10% with decreases of >2.5-fold for GAP43 and >3.5-fold for neurofilament. Semaphorin 3A protein expression was detected in NC tissue (Fig. 1D).

Conclusion

NCT inhibited the percentage of neurite-expressing SH-SY5Y cells including mean neurite length suggesting that NC produce soluble factors which may limit nerve ingrowth into the disk. This is supported by decreases in the expression of neuronal differentiation and outgrowth markers such as GAP43. Assessment of cell viability demonstrated that CM was not cytotoxic suggesting that NC soluble factors may be effective at inhibiting growth without neural-associated cytotoxic effects to the spinal cord and nerve roots. Significant increases in cell number were observed at doses of 10 and 50% but not at 100%. This nonlinear dose response suggests that NCT, while containing factors that can inhibit neuronal differentiation and outgrowth and promote cell proliferation, acts with different effects at different doses, emphasizing the need for mechanistic studies. We identified expression of neural inhibiting protein semaphorin 3A in NC tissue which was used to produce NCT; thus blocking studies to explore semaphorin 3A as a potential mechanism for inducing such neurite inhibiting effects is of key importance. We conclude that NCT contains soluble factors that inhibit neural growth and may have potential to slow or reverse painful disk degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Freemont, et al. Lancet 1997 Freemont, et al. J Path 2002 Keynes, et al. Neuron 1997 Larsson, et al. Spine 2011 Moon, et al. Spine 2012