Fig. 5: Quantitative imaging protocols for monitoring spatiotemporal changes in cytoskeletal structure during stem cell adaptation to cues driving proliferation and differentiation.

Live cell, fixed cell, and combined imaging methods have proven to be useful in monitoring cytoskeletal changes indicative of lineage commitment as a function of time, with time resolution from within minutes to hours (live cell imaging), and or days, e.g. 24 h (fixed cell imaging). a Dynamic changes in actin and tubulin cytoskeleton (quantified as filament length (b) and volume (c)) are observed as early as 15 min during neural differentiation of induced pluripotent stem cells (iPSCs)-derived neurons via time-lapse imaging¹⁴⁴. Simultaneous imaging and introduction of volume-changing stress (seeding density) and shape-changing stress (flow)^39,82 (d) enable elucidation of spatiotemporal cytoskeletal adaptation to controlled mechanical cues. Spatial distribution of actin (e) and tubulin (f) are measured at 30 min intervals as fluorescence intensity within the thickness of the cell and the total cell volume⁶. g Combining live and fixed cell labeling and imaging of actin using fluorescent-conjugated SiR-actin (SA) and phalloidin, respectively, SiR-actin-based measurement of actin turnover (SMAT) analysis distinguishes MSC differentiation (h) toward adipogenic (red line), chondrogenic (green line), and osteogenic (blue line) lineages, at timepoints as early as 1 h. Reduction in probe intensity (i) is observable within a few hours of switching from adipogenic induction medium (AD) to basal medium (BA), as shown qualitatively (j)¹¹⁵. Actin cytoskeleton morphometric descriptors, including shapes, intensities, and spatial distribution, represent the apparent changes that are readily detected within 24 h of hMSC differentiation. k The lineage commitment propensity of hMSCs cultured in respective differentiation induction media on various substrates could be parsed using descriptor-based computational modeling. The resulting confocal images are processed using Gaussian filter, enhanced and segmented for each single cell (l) to generate 43 descriptors (m). n Multidimensional scaling reduces the combination of descriptors into 3D space in a nonlinear fashion. Scatter plots in this 3D space show clear, time-dependent segmentation of adipogenic and osteogenic differentiation on non-treated glass (o–r), which can be observed after 72 h (q)¹⁴⁷. Microtubules mediate nuclear deformations, invagination, volume increase (s, t) through imposing constraints on the swelling nucleus during hPSC early differentiation into myeloid progenitors. This can be monitored within 24–72 h and quantified via 3D reconstruction of confocal images¹²⁰.