Abstract
Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (∼5 s) and most probable minimum distance of approach (4–6 µm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (∼18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and “stemness”. Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion.
Highlights
Stem cells are found in many tissues; they possess the unique ability to self-renew and differentiate into multiple cell types, properties that enable them to play a vital role in the maintenance of tissue integrity and homeostasis as in repair subsequent to tissue damage
Time–lapse imaging of neurosphere cultures have shown neural progenitor/stem cells (NSCs) and neursopheres to be highly motile, dynamic structures that tend to aggregate even at low cell densities [2,7]; these structures show intrinsic, spontaneous locomotion, propelled in part by tiny beating cellular surface processes when left untouched in incubators [2], and they frequently fuse when moved during observation by the experimenter [6], producing an inherent error in the neurophere assay in terms of clonality, size and number of neurospheres
To probe the temporal dynamics of these processes the interaction time between NSCs was varied and it was observed that, in all instances, a most probable minimum interaction time of,5 s was necessary for irreversible adhesion to occur
Summary
Stem cells are found in many tissues; they possess the unique ability to self-renew and differentiate into multiple cell types, properties that enable them to play a vital role in the maintenance of tissue integrity and homeostasis as in repair subsequent to tissue damage. The lack of specific phenotypic cell expression markers and access to fairly pure populations of stem cells has necessitated the use of functional assays to study neural stem cells in vitro and their potential to be evaluated by transplantation in vivo [1]. Stem cells and their progenitors are typically cultured in vitro either as dissociated two-dimensional adherent monolayers or three-dimensional neurospheres in suspension. Time–lapse imaging of neurosphere cultures have shown NSCs and neursopheres to be highly motile, dynamic structures that tend to aggregate even at low cell densities [2,7]; these structures show intrinsic, spontaneous locomotion, propelled in part by tiny beating cellular surface processes when left untouched in incubators [2], and they frequently fuse when moved during observation by the experimenter [6], producing an inherent error in the neurophere assay in terms of clonality, size and number of neurospheres
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