Nanoscale Molecular Quantification of Stem Cell-Hydrogel Interactions

Abstract

A common approach to tailoring synthetic hydrogels for regenerative medicine applications involves incorporating RGD cell adhesion peptides, yet assessing the cellular response to engineered microenvironments at the nanoscale remains challenging. To date, no study has demonstrated how RGD concentration in hydrogels affects the presentation of individual cell surface receptors. Here we studied the interaction between human mesenchymal stem cells (hMSCs) and RGD-functionalized poly(ethylene glycol) hydrogels, by correlating macro- and nanoscale single-cell interfacial quantification techniques. We quantified RGD unbinding forces on a synthetic hydrogel using single cell atomic force spectroscopy, revealing that short-term binding of hMSCs was sensitive to RGD concentration. We also performed direct stochastic optical reconstruction microscopy (dSTORM) to quantify the molecular interactions between integrin α5β1 and a biomaterial, unexpectedly revealing that increased integrin clustering at the hydrogel-cell interface correlated with fewer available RGD binding sites. Our complementary, quantitative approach uncovered mechanistic insights into specific stem cell-hydrogel interactions, where dSTORM provides nanoscale sensitivity to RGD-dependent differences in cell surface localization of integrin α5β1. Our findings reveal that it is possible to precisely determine how peptide-functionalized hydrogels interact with cells at the molecular scale, thus providing a basis to fine-tune the spatial presentation of bioactive ligands.

Keywords: AFM; PEG hydrogel; RGD; dSTORM; integrin α5β1; single cell force spectroscopy.

Figure 1

Schematic of stem cell-hydrogel interfacing. (a) 8-arm PEG-norbornene (20 kDa) is cross-linked with nondegradable PEG-dithiol (1000 Da), with tethered 6.8 mM cell-adhesive RGD peptide (CGGRGDSP) or nonadhesive RDG scrambled peptide (CGGRDGSP), in the presence of photoinitiator and 365 nm light to generate a 3D photo-cross-linked hydrogel network. (b) hMSCs bound to an AFM tip are brought into contact with hydrogels of varying RGD concentration, permitting analysis of specific unbinding events by single cell force spectroscopy (SCFS). (c) hMSCs attach to functionalized cantilevers through concanavalin-A—cell membrane glycoprotein interactions. Scale bar = 30 μm. (d) hMSCs adhered to hydrogels are fixed and immunolabeled, and surface localization of the RGD binding integrin α5β1 is imaged using the super-resolution imaging technique direct stochastic optical reconstruction microscopy (dSTORM). (e) Integrins in their active, extended conformation are immunolabeled with a primary antibody and visualized by detecting blinking of secondary antibody-bound AlexaFluor647.

Figure 2

Migration analysis of hMSCs on RGD hydrogels. (a) Representative confocal images of hMSCs bound to hydrogels, labeled for actin (green) and nuclei (blue). Scale bar = 200 μm main image, 20 μm inset. (b) Representative images of CellTracker Orange-labeled hMSC trajectories on the hydrogels, tracked over 6 h. Scale bar = 200 μm. (c) Migration directionality plots of hMSCs. N = 71 tracks per condition. (d) Average velocity of hMSCs on hydrogels with different RGD concentration. N = 748–1203 tracks from n = 3 hydrogel replicates per condition. (e) Total displacement of hMSCs on hydrogels. N = 748–1353 cells from n = 3 hydrogel replicates per condition. Nonparametric two-tailed t test with Mann–Whitney post hoc test. Violin plots represent median ± IQR. ***p < 0.001.

Figure 3

Single cell force spectroscopy of hMSCs detaching from the hydrogel surface. (a) Representative force–distance retraction curve when the hMSC-functionalized AFM tip is retracted from the surface of 100% RGD hydrogel. Typically, there is a large initial nonspecific adhesion of the hMSC to the hydrogel, followed by smaller force events after the bulk of the hMSC has detached as it moves away from the surface. The integrated area under the curve (blue) represents the total adhesion energy binding the single hMSC to the hydrogel surface. Inset illustrates ramp-like change in force (dotted line) preceding a force step event (between solid lines). (b) Total adhesion energy for hMSCs bound to 100%, 10%, and 0% RGD hydrogels. n = 7–13 per condition. Parametric one-way ANOVA with Tukey multiple comparison test. Box plots represent median ± IQR, whiskers represent minimum and maximum. ns = not significant. (c) Histogram of total registered force events during unbinding of hMSCs from 100% and 10% RGD hydrogels. N = 3 cells measured at n = 5 locations per n = 3 hydrogel replicates per condition. (d) Average total and RGD specific rupture events occurring on 100% and 10% RGD hydrogels, as defined through controlled interaction experiments (see Figure S5). N = 3 cells measured at n = 5 locations per n = 3 hydrogel replicates per condition. (e) Percentage of RGD rupture events per force displacement curve occurring on 100% and 10% RGD hydrogels.

Figure 4

dSTORM imaging of integrin α5β1. (a) Schematic representing immunolabeling of active integrin α5β1. (b) Representative dSTORM reconstructed images of integrin α5β1 clusters on the surface of hMSCs bound to 100% and 10% RGD hydrogels. Scale bar = 500 nm. (c) Representative DBSCAN cluster maps of images in panel b. Maps are 4 × 4 μm. (d) Representative cluster density maps of images in panel b. Maps are 4 × 4 μm. (e) Analysis of total number of surface localizations, number of clusters and density of clusters of integrin α5β1 per ROI for hMSCs in contact with 100% and 10% RGD hydrogels. N = 10–15 ROIs from n = 3 hydrogel replicates per condition. Welch’s unequal variances unpaired two-tailed t test. Box plots represent median ± IQR; whiskers represent minimum and maximum. *p < 0.05.

Figure 5

Correlation of multiscale interfacing parameters. 2D Radar plot of median measurements on 100% and 10% RGD hydrogels, with normalized values indexed between 0 and 1 on each axis. Migration velocity was indexed between 0 and 3 μm/min; adhesion energy was indexed between 0 and 600 nJ. Total rupture events were indexed between 0 and 12, and RGD rupture events were indexed between 0 and 5. Total localizations were indexed between 0 and 1500 per ROI, and the number of clusters were indexed between 0 and 100 per ROI.