Dimensionality and spreading influence MSC YAP/TAZ signaling in hydrogel environments

Abstract

Improved fundamental understanding of how cells interpret microenvironmental signals is integral to designing better biomaterial therapies. YAP/TAZ are key mediators of mechanosensitive signaling; however, it is not clear how they are regulated by the complex interplay of microenvironmental factors (e.g., stiffness and degradability) and culture dimensionality. Using covalently crosslinked norbornene-functionalized hyaluronic acid (HA) hydrogels with controlled stiffness (via crosslink density) and degradability (via susceptibility of crosslinks to proteolysis), we found that human mesenchymal stem cells (MSCs) displayed increased spreading and YAP/TAZ nuclear localization when cultured atop stiffer hydrogels; however, the opposite trend was observed when MSCs were encapsulated within degradable hydrogels. When stiffness-matched hydrogels of reduced degradability were used, YAP/TAZ nuclear translocation was greater in cells that were able to spread, which was confirmed through pharmacological inhibition of YAP/TAZ and actin polymerization. Together, these data illustrate that YAP/TAZ signaling is responsive to hydrogel stiffness and degradability, but the outcome is dependent on the dimensionality of cell-biomaterial interactions.

Keywords: Degradation; Dimensionality; Hydrogel; Mechanotransduction; Stiffness; YAP/TAZ.

Fig. 1. NorHA hydrogels enable independent tuning of substrate stiffness, degradability, and dimensionality

(a) Hyaluronic acid modified with norbornenes (~ 20 wt% of disaccharide repeat units) that permit (b) UV light-mediated thiol-ene addition reactions between thiolated RGD to permit cell attachment (2 mM final concentration) and with dithiol peptide crosslinkers (protease-degradable: GCNSVPMS↓MRGGSNCG, non-degradable: GCHGNSGGSGGNEECG) in the presence of the photoinitiator lithium acylphosphinate (LAP). Hydrogels (4 wt%) were fabricated as either thin films for seeding of MSCs atop for 2D culture or as plugs for MSC encapsulation within for 3D studies, across a range of crosslink densities (Low, Medium, High). AFM and DMA were used to quantify the elastic moduli of (c) 2D hydrogel thin films and (d) 3D hydrogel plugs, respectively (n = 3 hydrogels per group, error bars represent s.e.m.). **: P < 0.01, ***: P < 0.001.

Fig. 2. MSC spreading is dependent on substrate stiffness and dimensionality

(a) MSC spread area and cell shape index when seeded atop hydrogels of varying stiffness (Low (1 kPa), Medium (5 kPa), High (20 kPa)). (b) Representative F-actin (red) and nuclear (blue) staining of cells cultured atop hydrogels (2D). (c) MSC volume and cell shape index when encapsulated within hydrogels of varying stiffness (Low (1 kPa), Medium (5 kPa), High (20 kPa)). (d) Representative F-actin (red) and nuclear (blue) average projections of cells encapsulated within hydrogels (3D). n > 40 cells per group. Scale bars: 50 µm. ***: P < 0.001.

Fig. 3. MSC YAP/TAZ nuclear localization is differentially regulated by hydrogel stiffness in 2D and 3D cultures

(a) YAP/TAZ nuclear localization for MSCs cultured atop hydrogels of varying stiffness (Low (1 kPa), Medium (5 kPa), High (20 kPa)) reported as mean values, as well as single cell scatter plots of YAP/TAZ nuclear intensity ratio as a function of spread area, cell shape index, and aspect ratio (b) Representative YAP/TAZ staining (green) of cells cultured atop of hydrogels (2D). Dashed white lines: nuclear outlines. (c) YAP/TAZ nuclear localization for MSCs encapsulated within hydrogels of varying stiffness (Low (1 kPa), Medium (5 kPa), High (20 kPa)) reported as mean values, as well as single cell scatter plots of YAP/TAZ nuclear intensity ratio as a function of volume, cell shape index, and aspect ratio. (d) Representative YAP/TAZ (green) average projections of cells encapsulated within hydrogels (3D). Dashed white lines: nuclear outlines. n > 40 cells per group. Scale bars: 50 µm. ***: P < 0.001.

Fig. 4. Degradable hydrogel environments significantly enhance YAP/TAZ nuclear translocation in 3D cultures

MSC (a) volume, (b) shape index, and (c) aspect ratio when cultured in stiffness-matched (Medium (5 kPa) formulation) non-degradable and degradable hydrogels after 7 days. (d) Representative F-actin (red) and nuclear (blue) average projections of cells encapsulated within stiffness-matched non-degradable and degradable hydrogels. MSC YAP/TAZ nuclear localization (e) quantification and (f) staining (green) in stiffness-matched (Medium (5 kPa) formulation) non-degradable and degradable hydrogels after 7 days. Dashed white lines: nuclear outlines. Single cell scatter plots of YAP/TAZ nuclear intensity ratio as a function of (g) volume, (h) cell shape index, and (i) aspect ratio. Medium degradable data reproduced from Fig. 2, 3 for comparison. n > 40 cells per group. Scale bars: 50 µm. ***: P < 0.001.

Fig. 5. MSC spreading in 3D degradable environments is YAP/TAZ and actin-dependent

MSC (a) volume, (b) shape index, and (c) aspect ratio when cultured in Medium (5 kPa) degradable hydrogels after 7 days with no inhibitor, YAP/TAZ inhibitor verteporfin (VP (+)), or actin polymerization inhibitor cytochalasin D (CytoD (+)). (d) Representative F-actin (red) and nuclear (blue) average projections of cells encapsulated in Medium (5 kPa) degradable hydrogels cultured in media with no inhibitor, VP (+), or CytoD (+). MSC YAP/TAZ nuclear localization (e) quantification and (f) staining (green) in degradable hydrogels after 7 days with no inhibitor, VP (+), or CytoD (+). Dashed white lines: nuclear outlines. Single cell scatter plots of YAP/TAZ nuclear intensity ratio as a function of (g) volume, (h) cell shape index, and (i) aspect ratio. Medium degradable data reproduced from Fig. 2, 3 for comparison. n > 40 cells per group. Scale bars: 50 µm. ***: P < 0.001.

Fig. 6. Summary of relationships between hydrogel stiffness and degradation with respect to YAP/TAZ signaling of MSCs in both 2D and 3D

(a) Increased hydrogel stiffness promotes increased spreading and YAP/TAZ nuclear localization for MSCs cultured atop hydrogels 2D cultures; however, increased crosslinking results in decreased spreading and YAP/TAZ signaling for MSCs encapsulated within hydrogels for 3D cultures. The increased crosslinking in 3D hydrogels presents a greater number of crosslinks that need to be cleaved for spreading. (b) A degradable hydrogel environment is necessary for cell spreading and YAP/TAZ nuclear translocation in 3D cultures, since stiffness-matched non-degradable hydrogels limited both spreading and YAP/TAZ signaling. (c) Small molecule inhibition of YAP/TAZ or actin polymerization alters how MSCs interact with hydrogel environments by reducing spreading.