Interplay of matrix stiffness and protein tethering in stem cell differentiation

Abstract

Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

Fig 1. Influence of substrate porosity on ASC differentiation

a, Elastic modulus measured via AFM (n = 3) for the indicated acrylamide:bis-acrylamide ratios. b, SEM images of PA hydrogels made with varying monomer to crosslinker ratios as indicated (scale bars, 50 μm [top and bottom], 10 μm [middle]). c, Alkaline phosphatase staining of ASCs on 13 and 30 kPa hydrogels of the indicated compositions after 14 days of culture in normal media. Arrowheads indicate stained but yet negative cells (scale bar, 500 μm). d, Oil Red O staining of ASCs on 4 kPa and 30 kPa hydrogels of the indicated compositions after 7 days of culture in adipogenic induction media. Arrowheads indicate stained but yet negative cells (scale bar, 100 μm). e, Displacement maps of embedded fluorescent particles resulting from ASC traction forces on 4 kPa and 30 kPa hydrogels of the indicated compositions (scale bar, 50 μm). f, Quantification of mean displacement was plotted for hydrogels of the indicated composition and stiffness range (n > 20; mean ± S.E.M.; N.S. = not significant).

Fig 2. Influence of protein tethering on ASC differentiation

a, Schematic depicting the interaction between an AFM tip (orange) functionalized with a collagen I antibody (C2456; green) and the hydrogel (blue) functionalized with bound collagen I (red). The black arrow indicates the direction of motion. A rupture event occurs following retraction of the tip from the surface. b, Measured rupture force (left) and rupture length (right) for rupture events that occurred on 10/0.3 30 kPa hydrogels activated with the indicated sulfo-SANPAH (SS) and collagen I concentrations (n = 500; mean ± S.E.M.; **p < 0.0001). c, Rupture length was measured for rupture events that occurred on 30 kPa hydrogels with indicated monomer to crosslinker ratios. Hydrogels were activated with either 0.2 mg/ml or 1 mg/ml sulfo-SANPAH. (n = 500; mean ± S.E.M.; **p < 0.0001). d, Images of ASCs stained for ALP expression on 10/0.3 hydrogels as a function of sulfo-SANPAH concentration after 14 days of culture in normal media (scale bar, 500 μm). e, Displacement maps of embedded fluorescent particles resulting from ASC traction forces on 10/0.3 hydrogels for a range of indicated sulfo-SANPAH concentrations (scale bar, 50 μm). f, Quantification of mean bead displacement for the indicated hydrogel stiffness and composition as well as sulfo-SANPAH (SS) concentration (n = 20; mean ± S.E.M; **p < 0.0001). g, Measured fibronectin FRET intensity ratio for ASCs on 4, 14, and 30 kPa hydrogels activated with the indicated concentrations of sulfo-SANPAH (n = 8; *p<0.05). h, Proposed model of a cell on a protein coated substrate attached to a rigid base (glass coverslip) where cell forces are translated through the protein and through the substrate. Deformations of the substrate are measured via TFM and deformations of the protein are measured via FRET.

Fig 3. Direct incorporation of a short adhesive peptide to the PA substrate

a, Elastic modulus measured via AFM (n = 3; N.S. = not significant). b, aPEG-RGD-dye incorporation is detected under UV light. c, SEM images of PA hydrogels of indicated stiffness made with varying RGD concentration (scale bar, 50 μm). d, Measured rupture force (left), number of events (middle), and rupture length (right) for rupture events that occurred on 10/0.3 30 kPa hydrogels coated with PEG-biotin (n = 1000; mean ± S.E.M.; N. S. = not significant; **p<0.0001). e, ALP staining of ASCs on 13 kPa and 30 kPa hydrogels with low, medium, and high concentrations of RGD (scale bar, 500 μm). f, ALP staining of ASCs on 30 kPa hydrogels of varying monomer to crosslinker ratio and constant high concentration of RGD after 14 days of culture in normal media (scale bar, 500 μm). g, Representative displacement map (left) of embedded fluorescent particles resulting from ASC traction forces on a 30 kPa hydrogel with 2.5 mM RGD. Mean displacement is shown (right) for a collagen coated hydrogel (0.2 mg/ml sulfo-SANPAH and 50 μg/mL collagen I) and a PA-PEG-RGD hydrogel (2.5 mM RGD). (n = 30; mean ± S.E.M; N.S. = not significant; scale bar, 50 μm).

Fig 4. Cells sense by contracting against their substrates

a, Schematic depicting how cells dynamically deform soft and stiff substrates by pulling (and not pushing) via myosin contractions. Softer substrates are deformed to a greater extent than stiffer substrates. b, Schematic depicting the interaction between an AFM tip and the surface of a substrate. The arrow indicates the direction of motion of the tip during retraction. c, Representative retraction curves for 1 and 30 kPa PA hydrogels and a 100:1 PDMS substrate. The dashed line indicates the point at which the tip is no longer indenting into the substrate (shaded light blue). Substrate stiffness is calculated by fitting the linear region of the retraction curve starting at the (undeformed) surface. d, Substrate spring constant determined by the method depicted in c for 1 and 30 kPa PA hydrogels and 50:1 and 100:1 PDMS substrates as a function of AFM tip retraction speed. Cells are sensitive to substrate stiffnesses of 1–100 kPa. Previously measured myosin contraction speeds range from 10–100 nm/s (gray). e, Alkaline phosphatase staining of ASCs on PDMS substrates after 7 days of culture in normal media (scale bar, 500 μm).

Fig 5. Atomic force spectrography analysis of ligand coated PDMS substrates

a, Measured rupture length (top) and rupture force (bottom) for rupture events detected on 50:1 PDMS substrates activated with the indicated sulfo-SANPAH (SS) and collagen or NH₂-PEG-biotin concentrations. No data indicates that no rupture events were detected. b, Percentage of spectrograms with at least one rupture event on PA and PDMS substrates activated with the linkers and ligands shown.