The effect of actin disrupting agents on contact guidance of human embryonic stem cells

Abstract

Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600nm ridges with 600nm spacing and 600+/-150nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and alpha-tubulin, and reduced proliferation. Spatial polarization of gamma-tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography.

Figure 1. Morphological and proliferative response of hESCs to nanotopography

A) Phase images of hESCs cultured on flat and linear nanotopograhy PDMS substrates. B) Circularity and average angle of alignment (all graphical data is reported as mean +/− s.d.) were reduced on nanotopographic substrates when compared to flat substrate. C) Cells cultured on nanotopographic substrates exhibited lower projected cell areas as well as determined by t-test (*** p < 0.001). D) Cell growth kinetics showed that cells cultured on nanotopography had higher initial cell densities followed by stagnant proliferation. These kinetics were in contrast to those of cells grown on flat surfaces, which exhibited continued proliferation through 120 h. E) Representative color micrographs of BrdU assay revealed increased uptake of hESCs cultured on flat substrates compared to cells cultured on nanotopographic substrates after 24 h. F) BrdU uptake was reduced from 24 h to 48 h across both types of substrates. Statistical significance across 24 h and 48 h timepoints was determined by two-way ANOVA (* p < 0.05). Scale bars in all micrographs are 100 μm.

Figure 2. Nanotopography induces organization of cytoskeleton proteins

Human ESCs cultured on substrates with linear nanotopographic features responded to topography and exhibited organized cytoskeleton proteins including: α-SMA, α-tubulin, vimentin, and calponin. These morphological alterations were also confirmed through SEM micrographs. Direction of grid lines is indicated by white arrow. Scale bars are 10 μm.

Figure 3. Deconvolution images of α-SMA in hESCs cultured on flat and nanotopographic substrates

Deconvolution images revealing α-SMA in hESCs cultured on flat substrates are globular without organization within the cytoskeleton (left panel). This observation lies in stark contrast to hESCs cultured on nanotopographic substrates (right panel) which exhibit organized aligned fibers in the direction of the nanotopography (right panel is a composite image of two micrographs). Direction of grid lines is indicated by white arrow. See online version for video of micrograph sections. Scale bars are 10 μm.

Figure 4. Nanotopography polarizes spatial distribution of gamma-tubulin complexes (GTCs) within hESCs

GTCs present in hESCs after 24 h of culture on flat substrates exhibit a random radial distribution with respect to the longitudinal axis of the nucleus when compared to cells cultured on nanotopographic substrates. Individual GTCs in each cell are indicated by white arrowheads. See Supplementary Fig 1 for additional information regarding the scoring of GTC alignment. Scale bars in all figures are 10 μm.

Figure 5. Actin-disrupting agents reduce nanotopography-mediated responses

Treatment of hESCs cultured on nanotopographic substrates, with cyto D, or latr B, two varieties of cytoskeleton disrupting agents, leads to a loss of cytoskeleton protein organization including α-SMA, vimentin, and α-tubulin. Culturing hESCs in the presence of ADAs also leads to a more rounded three-dimensional cell morphology. Scale bars are 10 μm.

Figure 6. Actin disrupting agents reduce morphological, proliferative, and metabolic effects of nanotopography-induced contact guidance

A) Morphological characterization of cells cultured on nanotopographic substrates in the presence of cyto D and latr B resulted in increased circularity and a loss of bias of alignment angle as confirmed by one-way ANOVA (*** < p 0.001). B) Increased cell densities at time points 24 h, and 48 h as confirmed by two-way ANOVA (*** p < 0.001) in combination with increased BrdU uptake at 24 h as measured by one-way ANOVA (*** p < 0.001) (C) suggests that treatment with actin-disrupting agents leads to increased proliferation. D) Metabolic activity, as measured by XTT cleavage assay, increased for cells cultured with treatment of both cyto D (i) and latr B (ii) treatment at 24 h when compared to no drug as measured by t-tests (* p < 0.05, * p < 0.05 for cyto D and latr B, respectively).