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 & Quantification

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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.

 
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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.
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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.

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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).