Live Imaging of Cortical Neural Stem Cell Neurite Outgrowth with IncuCyte NeuroTrack
Experimental Objectives
1) Differentiation and quantification:
To perform a morphological characterization of Axol Cortical Neural Stem Cells throughout the differentiation process and understand the suitability of Essen's phase contrast NeuroTrackTM analysis algorithm.
Method development (cell density, coating conditions, cell feeding protocol).
2) Passaging post-differentiated cells:
To assess the possibility of passaging the cells post plating to create a model to study both neurite outgrowth as well as neurodegeneration.
3) Cell Type Comparison:
To complete a time-course comparison of neurite outgrowth using N2a cells, Primary cells, Competitor iPSC-derived neurons and Axol Cortical Neural Stem Cells-derived Neurons.
Differentiation of Axol Cortical Neural Stem Cells
Axol cortical NSCs were successfully differentiated into human cerebral cortical neurons following treatment with Axol Neural AdvanceTM.
Differentiation & Quantification Analysis Algorithm – Masking of cells
Fast differentiation process
High number of quantifiable neurites
Neurite Length is dependent on seeding density
The highest neurite length is observed with a seeding density of 10,000 cells per well.
Passaging of Post-Differentiated Cells
Cells were defrosted into 35-mm dish and differentiated for 4 days as per protocol.
Cells were lifted with Axol UnlockTM and seeded into 1x 96-well TPP plate in Axol Sure BoostTM.
24 h post-plating 100 % of the media was replaced.
Neurites establishment occurred within 6 h post-plating.
Cell Type Comparison
Time-courses compare the mean ± SEM of NL, BP and NL/CBC values for rat primary cortical neurons, competitor iPSC-derived neurons, Axol-hNPC-derived neurons and N2a cells.
High levels of neurites can be quantified using Zoom algorithm (60 - 70 mm/mm2, compared to 25 – 30 mm/mm2 for competitor iPSC-derived neurons).
Overall, Axol NSCs yielded high Neurite length and branch point values when compared to any other cell type.
Conclusions
Differentiated Axol NSCs can be passaged with a high survival rate.
Onset of neurite outgrowth occurs within 6 h post passaging.
NSCs yielded high Neurite length and branch point values when compared to competitor iPSC-derived neurons, primary rat cortical neurons or N2a cells (~ 60, ~25, ~30, ~20 mm/mm2, respectively).
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