Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation

Abstract

Mesenchymal stem cell (MSC) therapy has demonstrated applications in vascular regenerative medicine. Although blood vessels exist in a mechanically dynamic environment, there has been no rigorous, systematic analysis of mechanical stimulation on stem cell differentiation. We hypothesize that mechanical stimuli, relevant to the vasculature, can differentiate MSCs toward smooth muscle (SMCs) and endothelial cells (ECs). This was tested using a unique experimental platform to differentially apply various mechanical stimuli in parallel. Three forces, cyclic stretch, cyclic pressure, and laminar shear stress, were applied independently to mimic several vascular physiologic conditions. Experiments were conducted using subconfluent MSCs for 5 days and demonstrated significant effects on morphology and proliferation depending upon the type, magnitude, frequency, and duration of applied stimulation. We have defined thresholds of cyclic stretch that potentiate SMC protein expression, but did not find EC protein expression under any condition tested. However, a second set of experiments performed at confluence and aimed to elicit the temporal gene expression response of a select magnitude of each stimulus revealed that EC gene expression can be increased with cyclic pressure and shear stress in a cell-contact-dependent manner. Further, these MSCs also appear to express genes from multiple lineages simultaneously which may warrant further investigation into post-transcriptional mechanisms for controlling protein expression. To our knowledge, this is the first systematic examination of the effects of mechanical stimulation on MSCs and has implications for the understanding of stem cell biology, as well as potential bioreactor designs for tissue engineering and cell therapy applications.

Fig. 1

Mechanical stimulation is applied individually in parallel from a single population of cells to determine a differential response to each of the stimuli

Fig. 2

a Average area measurements normalized by controls. b Changes in shape index relative to controls. c Cell density normalized by controls. The dashed line represents control values. All data are presented as mean +/− SEM. * denotes p < 0.05 compared to controls. ⁺ denotes p < 0.1 compared to controls. † denotes p < 0.05 for comparison of means within each stimulus. (1), (2), and (3) denote p < 0.05 for comparison of means between stimuli of the corresponding bar pattern where (1) = CS, (2) = CP, and (3) = LSS

Fig. 3

Normalized histograms for cellular orientation in MSCs presented as the mean +/− SEM. Non-parametric analysis against a uniform distribution resulted in no statistical significant differences with any of the cyclic pressure regimens, which is in agreement with the normalized histogram for the control. No statistical difference was found for LSS at 1 dyne/cm² and 5 dynes/cm². However, LSS at 10 dynes/cm² and 20 dynes/cm², a preferred orientation begins to develop around 90°, which is in the direction of flow. CS at 1% 2.75 Hz and 1 % 1 Hz did not demonstrate any significant changes in alignment. However, higher magnitudes of CS (5% 1 Hz and 10% 1 Hz) demonstrated a significant change from a uniform distribution centered on 90°, which is perpendicular to the direction of stretch

Fig. 4

MSCs exposed to CS expressed SMC proteins with increasing levels of differentiation with increasing magnitudes of stretch at 1 Hz. Increasing the frequency of stimulation also caused local expression of all SMC proteins assayed and may indicate a population-specific frequency response. Arrows indicate the direction of applied stimulation. Note qualitatively the effect of CS on cellular alignment

Fig. 5

Temporal changes in muscle-related gene expression for each component of the Mechanical Panel. CS-10 showed the more conisten trend toward upregulation by 72 h, despite several genes being down-regulated at 24 h. * p < 0.05; ⁺ p < 0.10

Fig. 6

Temporal changes in endothelial-related gene expression were significantly higher than the muscle-related genes. LSS-20 and CP-120 demonstrated more significant and sustained endothelial-specific genes than CS-10. * p < 0.05; ⁺ p < 0.10