Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue - PubMed (original) (raw)
Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue
D Adam Young et al. Biomaterials. 2013 Nov.
Abstract
Biochemical and biomechanical extracellular matrix (ECM) cues have recently been shown to play a role in stimulating stem cell differentiation towards several lineages, though how they combine to induce adipogenesis has been less well studied. The objective of this study was to recapitulate both the ECM composition and mechanical properties of adipose tissue in vitro to stimulate adipogenesis of human adipose-derived stem cells (ASCs) in the absence of exogenous adipogenic growth factors and small molecules. Adipose specific ECM biochemical cues have been previously shown to influence adipogenic differentiation; however, the ability of biomechanical cues to promote adipogenesis has been less defined. Decellularized human lipoaspirate was used to functionalize polyacrylamide gels of varying stiffness to allow the cells to interact with adipose-specific ECM components. Culturing ASCs on gels that mimicked the native stiffness of adipose tissue (2 kPa) significantly upregulated adipogenic markers, in the absence of exogenous adipogenic growth factors and small molecules. As substrate stiffness increased, the cells became more spread, lost their rounded morphology, and failed to upregulate adipogenic markers. Together these data imply that as with other lineages, mechanical cues are capable of regulating adipogenesis in ASCs.
Keywords: Adipose tissue engineering; Cell morphology; Soft tissue biomechanics; Stem cell.
© 2013 Elsevier Ltd. All rights reserved.
Figures
Figure 1. Morphological Changes Associated with Adipogenic Media
ASCs that were cultured in growth media (A) exhibited no lipid development compared to the significant lipid accumulation noted in cells cultured in adipogenic media (B) at Day 14. While no changes in cellular area were noted between the two experimental groups (C), the cells in adipogenic media did show a significantly reduced cell aspect ratio compared to those in growth media (D). Scale bar = 50 μm. * indicates p < 0.001 using a t-test to compare cells in growth media to those in adipogenic media.
Figure 2. Polyacrylamide Gels of Varying Stiffness
Polyacryalamide gels were produced by mixing 8% acrylamide monomer with varying concentrations of bis-acrylamide. AFM analysis revealed that the gels exhibited significantly increased elastic moduli as bis-acyrlamide concentration increased. * indicates p < 0.001 compared to all other concentrations.
Figure 3. Morphological Changes Associated with ASCs Cultured on Polyacrylamide Gels
ASCs were attached and viable on polyacrylamide gels with stiffnesses ranging from 2 kPa (A), 20 kPa (B), and 40 kPa (C), and compared to those on standard tissue culture plastic (D). When cultured in growth media, the ASCs displayed increasing cell spreading as the substrate stiffness increased (A–D, Green = F-actin, Blue = nuclei). Accordingly, cell area increased with increasing stiffness (E). Cell aspect ratio also increased with substrate stiffness, with ASCs on 40 kPa gels having the largest aspect ratio and ASCs on 2 kPa gels having a significantly reduced aspect ratio (F). Scale bar = 50 μm. * indicates p < 0.01 compared to all other substrates.
Figure 4. Soft Substrates Encourage Adipogenesis of ASCs
ASCs cultured in growth media for 6 days on 2 kPa gels (A) exhibited positive lipid accumulation, as identified by the red stain Oil Red O, compared to no staining observed for cells cultured on 20 kPa gels (B), 40 kPa gels (C), or tissue culture plastic (D). Examination of gene expression via PCR confirmed that ASCs on 2 kPa gels indeed expressed significantly upregulated levels of the adipogenic markers PPARγ, CEBPα, and aP2 compared to cells cultured on tissue culture plastic. Scale bar = 50 μm. * indicates p < 0.05 compared to tissue culture plastic control group.
Figure 5. Effect of Cytochalasin-D on Cell Morphology
Addition of the actin polymerization inhibitor, cytochalasin–D, caused the cells to adopt a more rounded morphology on 2 kPa (A), 20 kPa (B), and 40 kPa (C) gels and tissue culture plastic (D), and disrupted intracellular actin filaments (A–D, Green = F-actin, Blue = nuclei). Disrupting these actin filaments with cytochalasin–D resulted in significantly reduced cellular area for cells on 20 and 40 kPa gels compared to growth media values, but had no effect on ASCs on tissue culture plastic (E; black columns; cytochalasin–D media; grey columns: normal growth media). More importantly, however, it caused a significant reduction in cell aspect ratio for ASCs on 20 and 40 kPa gels and those cultured on standard tissue culture plastic compared to values in growth media (F, black columns; cytochalasin–D media; grey columns: normal growth media). There were no significant differences in aspect ratio between substrates in cytochalasin media. Scale bar = 50 μm. * indicates p < 0.001 compared to other groups in cytochalasin-supplemented media. # indicates p < 0.001 compared to same group in normal growth media.
Figure 6. Decreased Aspect Ratio Stimulates Adipogenesis
ASCs cultured in cytochalasin–D for 6 days exhibited a more rounded morphology and as a result, stained positively for lipid accumulation via Oil Red O (arrows) regardless of the underlying substrate stiffness of 2 kPa (A), 20 kPa (B), 40 kPa (C), or standard tissue culture plastic (D). Investigating the gene expression also revealed their adipogenic nature, as these cells exhibited significantly higher levels of all three adipogenic markers PPARγ, CEBPα, and aP2 compared to control cells cultured on tissue culture plastic in growth media. Scale bar = 50 μm. * indicates p < 0.05 compared to tissue culture plastic control group; # indicates p < 0.01 compared to all groups.
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