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Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay
Published on: January 12, 2011
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Assembling neurospheres: dynamics of neural progenitor/stem cell aggregation probed using an optical trap
Uma Ladiwala1, Himanish Basu, Deepak Mathur
1UM-DAE Centre for Excellence in Basic Science, University of Mumbai, Kalina Campus, Mumbai, India. brainwave@cbs.ac.in
Plos One
|June 14, 2012
Summary
Neural progenitor/stem cells (NSCs) require minimal time and distance for irreversible adhesion, quantified using optical trapping. Findings challenge the validity of the neurosphere assay for measuring stemness.
Area of Science:
- Biophysics
- Cell Biology
- Neuroscience
Background:
- Neural progenitor/stem cells (NSCs) aggregate to form neurospheres, a common method for assessing stemness.
- The precise biophysical mechanisms and temporal dynamics of NSC adhesion are not fully understood.
Purpose of the Study:
- To investigate the mechanics and spatio-temporal dynamics of neural progenitor/stem cell (NSC) adhesion and aggregation.
- To quantify the minimum time, distance, and force required for irreversible NSC adhesion.
- To evaluate the implications of these findings for the neurosphere assay.
Main Methods:
- Utilized optical trapping (tweezing) combined with fluid flow technology.
- Quantified filopodial- and membrane-mediated adhesion characteristics.
- Employed computational modeling for validation.
- Investigated the effects of calcium ion removal and F-actin disruption.
Main Results:
- Determined the minimum time (∼5 s) and distance (4-6 µm) for irreversible NSC adhesion.
- Quantified the lower limit of NSC adhesive force at approximately 18 pN.
- Observed oscillatory motion in filopodia-mediated adhesion post-aggregation.
- Found that calcium ion removal inhibits both filopodia- and membrane-mediated adhesion, while F-actin disrupts filopodia-mediated adhesion.
Conclusions:
- The neurosphere assay may not accurately reflect NSC clonality and stemness due to the irreversible nature of cell aggregation.
- Established biophysical parameters for NSC adhesion provide new insights into neural development and cell-cell interactions.
- Disassembly of aggregated neurospheres is difficult via mechanical or thermal means.
