Dynamic Surfaces for the Study of Mesenchymal Stem Cell Growth through Adhesion Regulation

Abstract

Out of their niche environment, adult stem cells, such as mesenchymal stem cells (MSCs), spontaneously differentiate. This makes both studying these important regenerative cells and growing large numbers of stem cells for clinical use challenging. Traditional cell culture techniques have fallen short of meeting this challenge, but materials science offers hope. In this study, we have used emerging rules of managing adhesion/cytoskeletal balance to prolong MSC cultures by fabricating controllable nanoscale cell interfaces using immobilized peptides that may be enzymatically activated to change their function. The surfaces can be altered (activated) at will to tip adhesion/cytoskeletal balance and initiate differentiation, hence better informing biological mechanisms of stem cell growth. Tools that are able to investigate the stem cell phenotype are important. While large phenotypical differences, such as the difference between an adipocyte and an osteoblast, are now better understood, the far more subtle differences between fibroblasts and MSCs are much harder to dissect. The development of technologies able to dynamically navigate small differences in adhesion are critical in the race to provide regenerative strategies using stem cells.

Keywords: dynamic cell/material interface; mesenchymal stem cell; metabolomics; stem cell growth.

Figure 1

Dynamic, enzyme cleavable surfaces for MSC growth. (A) Cartoon representation of the FMOC and PEG blocked RGD surfaces illustrating the elastase cleavage site. Plain glass coverslips were modified using silanization and PEGylation steps followed by solid-phase peptide synthesis to build up the full structure in a stepwise manner. The incorporation of a dialanine enzyme cleavable linker facilitates the removal of the FMOC/PEG blocking group, thereby forming the basis of the switch. (B) ToF-SIMS image shows a uniform chemical surface composition on the micron scale and confirms that PEG, FMOC, and amino acids (shown as the sum of the indicated representative peaks for each amino acid) are only detected in the analysis where expected; that is, FMOC only seen on the FMOC-D surface and amino acids was only noted when the AARGD sequence was present. Color scales represent ion counts. Images for specific ions are presented on the same scale for all samples; total ion images are scaled to their individual range. Note that inset images in the FMOC column are all similarly brightness-enhanced versions of the main images to show differences more clearly. (C) MSCs were cultured on plain glass coverslips for 48 h and treated with different concentrations of elastase ranging from 1.0 to 0.1 mg/mL (4.60 to 0.460 U) in basal cell culture medium. Live/dead stain for elastase tolerance showed that while cell adhesion was clearly affected at the higher concentrations, few remaining dead cells were noted with any elastase concentration (they may have detached). However, cells incubated with 0.1 mg/mL elastase were indistinguishable from controls, whereas the positive controls (ethanol addition) indicated uptake of the ethidium homodimer; thus these cells were dead. (D) Surface-bound FMOC groups were seen to fluoresce at a wavelength of 315 nm (left-hand spectrum), and piperidine cleavage resulted in a loss of this signal (middle spectrum). At 0.1 mg/mL, it was seen that elastase cleaved surface-bound FMOC (right-hand spectrum); n = 3.

Figure 2

Dynamic control of MSC adhesion and tension. (A) MSCs cultured at 7 cells/mm² were seen to spread to a greater degree on plain controls, RGD controls, FMOC-RGD (low), and cleaved RGD (high) surfaces and to a smaller degree on PEG₁₈ and all RGE surfaces, creating a pronounced cell size difference between the surfaces. As morphology was altered, adhesion and cytoskeletal arrangement also changed with RGD controls and cleaved RGD (high) surfaces, supporting more organized stress fibers and larger adhesions. Red = actin, green = vinculin, and blue = nuclei. (B) Adhesion subtypes were recorded as a percentage of the average number of adhesions identified per cell. The majority of adhesions were focal adhesions (FAs), with focal complexes (FXs) and supermature adhesions (SMAdhs) making up a much smaller percentage. As a whole, more FXs were observed per cell on plain, PEG₁₈, RGE controls, FMOC-RGD (low), and cleaved RGE substrates, while more SMAdhs were observed on RGD controls and cleaved RGD (high) surfaces. In line with differences in cell size and adhesion length, p-myosin expression, as a measure of cytoskeletal tension, showed the cells were under increased tension on RGD controls and cleaved RGD (high) surfaces. Error bars are standard error of the mean; stars indicate significant difference between groups as determined by one-way ANOVA and Dunn’s post-hoc test, where *P < 0.5, **P < 0.01, and ***P < 0.001 - § = plain/PEG different to RGD by P < 0.05; n = 40 cells per substrate and three material replicates.

Figure 3

Integrin, BMP2 receptor, and cytoskeletal tethering changes in MSCs on dynamic surfaces. (A) Integrin β₁ and BMPR1a staining in MSCs cultured on FMOC-RGD (low) at t₀ (t₀ is after 48 h of culture immediately before addition of elastase) and then after 24 and 48 h post-elastase treatment (cleaved RGD) or in FMOC-RGD surfaces. β₁ was observed to be found in punctate adhesions, with little BMPR1a colocalization noted (48 h inset). On the cleaved RGD (high) surfaces, BMPR1a was seen with adhesion morphology but in different areas to the regions of β₁ localization (48 h outset). (B) Integrin β₅ and BMPR1a staining in MSCs on FMOC-RGD surfaces at t₀ (immediately before addition of elastase) and then at 24 and 48 h post-elastase (cleaved RGD) or in FMOC-RGD surfaces. On the FMOC-RGD surfaces, little β₅ expression and no BMPR1a colocalization were observed. However, on the cleaved surfaces, strong β₅/BMPR1a colocalization was noted by 24 h (outset images). (C) Actin/ezrin colocalization could be seen at t₀ and on FMOC-RGD. However, for cleaved RGD samples, ezrin appeared to colocalize with cortical actin at the cell periphery (arrows). SiRNA knock-down of ezrin resulted in an increase in pRUNX 2 5 days post-switch. For pRNUX2, in-cell western analysis, n = 3, results are mean ± SD, stats by ANOVA and Dunn’s post-hoc test where *P < 0.05.

Figure 4

Analysis of MSC growth and differentiation at days 7 and 21. (A) Immunofluorescence images of STRO-1 MSCs at day 21. At this time point, the STRO-1 marker had substantially decreased on all surfaces, remaining only on plain controls and FMOC-RGD (low) surfaces (outset images). ALCAM was still easily detectible on the plain controls and FMOC-RGD but reduced on RGD controls and cleaved RGD (high) surfaces. OPN and OCN levels were increased on RGD controls and cleaved RGD with respect to the other surfaces. Red = actin, green = STRO-1/ALCAM/OPN/OCN, and blue = nuclei; scale bar is 100 μm. (B) Graphs show quantification of STRO-1 expression at 7, 14, and 21 days of culture. At day 7, almost all cells on plain controls, FMOC-RGD, and cleaved RGD surfaces expressed STRO-1. This reduced with time until, at day 21, less that 15% of cells on plain controls and the cleaved RGD surface retained STRO-1 expression, while double this number, >30%, retained STRO-1 expression on FMOC-RGD (approximately 100 cells were included in the quantification; n = 2). (C) OCN quantification. Data represent OCN levels expressed by MSCs cultured on plain glass, RGD controls, FMOC-RGD, and cleaved RGD surfaces at days 7 and 21 of culture. OCN expression was observed to increase most on RGD controls and cleaved RGD compared to the other surfaces. (D) OPN quantification. Data represent OPN levels expressed by MSCs seeded on plain glass, RGD controls, FMOC-RGD, and cleaved RGD at days 7 and 21 of culture. OPN expression was observed to increase most on the RGD controls and cleaved RGD surfaces. Statistics carried out by one-way ANOVA and Dunn’s post-hoc test, where *P < 0.05, **P < 0.01, and ***P < 0.001, n = 3. Note that au = arbitrary units.

Figure 5

Metabolite analysis of MSCs on dynamic surfaces. (A–C) Putative metabolites were analyzed using MetaboAnalyst 2.0, and the data were displayed as volcano plots relative to D2-plain (plain control at day 2). The y-axis refers to the p value (determined by two-tailed t test), with the x-axis intercept set at P = 0.05 so that all data points above the x-axis represent metabolites that were significantly different from controls. The x-axis represents fold change as a measure of the magnitude in difference between samples and the control. Data points to the left of the y-axis are metabolites down-regulated with respect to controls, and data points on the right of the y-axis were up-regulated (n = 3). (D) At 7 days (2 days “low” and 5 days “high” for cleaved RGD), principle component analysis showed very clear metabolomic differences with MSCs on the FMOC-RGD (low) surface having a highly homogeneous metabolome, more so than on the controls, and the cleaved RGD (high) surfaces having a far more heterogeneous metabolome (n = 3). (E) Heatmap of putatively detected unsaturated lipids after 7 days culture showing up-regulation in MSCs on the FMOC-RGD surface and down-regulation in MSCs on the cleaved RGD surface (n = 3). (F,G) Ingenuity functional pathway analysis illustrating more significantly altered functional pathways in MSCs on the cleaved RGD surface (F) compared to those on FMOC-RGD surfaces (G). Functions include carbohydrate, small molecule, nucleic acid, lipid and vitamin metabolism, cell growth and proliferation, and skeletal development pathways (statistics by Fischer’s exact test, P < 0.05 represented by bars higher than the threshold arrow, n = 3).

Figure 6

Analysis of MSC adhesion and differentiation on PEG blocked surfaces. (A) Immunofluorescence images of vinculin in adhesions at day 5 and STRO-1 at day 21 of culture. On the PEG-RGD (low) substrates, MSCs were observed to have smaller adhesions and increased expression of the STRO-1 MSC marker (arrows) compared to MSCs seeded on RGD cleaved surfaces (high) where, again, larger adhesions and loss of STRO-1 expression was observed. Red = actin, green = vinculin or STRO-1, and blue = nucleus. (B) Image analysis after 21 days of culture confirmed these results, demonstrating that more MSCs retained STRO-1 expression on PEG-RGD than cells on plain controls and cleaved RGD surfaces which typically lose STRO-1 much faster (results are mean ± SD, statistics by ANOVA, *P < 0.05). (C) Principle component analysis from metabolomics data illustrates most data homogeneity on PEG-RGD surfaces and most heterogeneity on the cleaved RGD surface as with the FMOC blocked samples (n = 3).