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Biomanufacturing

Biomanufacturing - Poster Session B

Poster B3 - Effects of Stiffness and Cell-Cell Mimetic Peptides on Stem Cell Phenotype and Matrix Mechanosensing

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Under the right conditions, mesenchymal stem cells (MSCs) can differentiate into cell types present in connective tissues including bone, cartilage, muscle, and fat. Although MSCs hold immense potential in tissue biofabrication applications, the FDA has yet to approve any MSC-based therapies in part due to the challenge in maintaining their naïve, undifferentiated phenotype through multiple passages. This is attributed to the use of supraphysiological stiff tissue culture plastic during many stem cell manufacturing workflows, and we hypothesize that softer materials with peptides that attenuate matrix mechanosensing will prolong stemness. To test our hypothesis, we designed norbornene-modified hydrogels with independently tunable stiffness and peptide-functionalization. Stiffness is negatively associated with stemness, and it was recently shown that N-cadherin cell-cell signaling reduces the ability for adherent cells to ‘sense’ material stiffness. HAVDI is an N-cadherin mimetic peptide, and to test our hypothesis we functionalized Soft and Stiff HANor hydrogels with these stiffness-attenuating peptides. To evaluate the effects of stiffness and HAVDI peptides on MSC mechanosensing, we cultured MSCs on top of these hydrogels and evaluated changes in cell shape (area, circularity) and the nuclear localization of well-established mechanosensitive proteins, YAP (Yes-associated protein) and RUNX2 (Runt-related transcription factor 2). In the absence of HAVDI, we expect to find that MSCs on Stiff hydrogels will be more spread (larger, less round), feature higher nuclear YAP/RUNX2, and that the inclusion of HAVDI will prevent stiffness-mediated matrix mechanosensing, which can help guide the design of softer, bioactive materials for manufacturing large populations of high quality MSCs.

Materials and

Methods:  Hydrogel formation: Hydrogels were formed by reacting norbornene-modified hyaluronic acid macromers (HANor) with di-thiolated crosslinkers, and pendant norbornenes in the hydrogel network were used to functionalize the hydrogels with mono-thiolated peptides (Figure 1A). HANor modification with norbornene groups was confirmed using 1H NMR. Soft and Stiff hydrogels were formed by photopolymerizing solutions (HANor 3 wt%, Irgacure 2959 0.05 wt%) that included low (0.4 mM) or high (2.0 mM) di-thiol crosslinker concentrations, respectively. Hydrogel stiffness was confirmed through compression testing. To incorporate bioactive signals, hydrogel solutions were supplemented with thiolated adhesive RGD peptide (2 mM, GCGYGRGDSPG) and thiolated cell-cell mimetic HAVDI peptide (2 mM, HAVDIGGGC) or scrambled, inactive HAVDI (2 mM, AGVGDHIGC) (Figure 1B). Hydrogel solutions were pipetted into 2D silicone molds (8 mm diameter, 0.5 mm height) and photopolymerized with ultraviolet light (10 mW/cm2, 10 minutes).

Cell seeding and analysis: Hydrogels were incubated in growth medium (αMEM, 10% fetal bovine serum, 1% penicillin/streptomycin) overnight, then MSCs (passage 3) were seeded on top (4,000 cells/cm2) for 24 hours. Samples were then fixed (10% formalin), permeabilized (0.1% Triton X-100), blocked (3% bovine serum albumin), and stained for YAP (Yes-associated protein, 1:250) or RUNX2 (Runt-related transcription factor 2, 1:250), actin filaments (AF568 phalloidin, 1:250), and nuclei (Hoechst, 1:1000). Samples were imaged with a confocal microscope, and single-cell analysis was performed using ImageJ. Nuclear YAP localization was quantified by taking the area-normalized ratio of nuclear and cytosolic YAP signal. Significance was established using one-way ANOVA followed by Tukey’s test for multiple comparisons.

Results, Conclusions, and Discussions: Results and

Discussion: 1H NMR spectra of HANor macromer shows that ~45% of hyaluronic acid repeat units are functionalized with norbornene (Figure 1C). Compression testing of Soft and Stiff HANor hydrogels resulted in average elastic moduli of 7.1 kPa and 13.0 kPa, respectively (Figure 1D). MSCs cultured on soft substrates typically remain small and circular compared to those cultured on stiff substrates. It is expected that cells cultured on Stiff hydrogels containing HAVDI will prevent cells from spreading, and will maintain a low nuclear to cytoplasmic ratio of YAP (Figure 2A). Representative images of single MSCs on Soft and Stiff hydrogels with and without HAVDI reflect these expectations (Figure 2B). Quantification of the results further validated the qualitative findings. MSCs on Stiff substrates experienced an increase in circularity and a decrease in cell area with HAVDI compared to MSCs on Stiff hydrogels without HAVDI. The average areas for cells cultured on Stiff gels with and without HAVDI were significantly different at 917 µm2 and 3,217 µm2, respectively (Figure 2C). The average cell circularity for Stiff HANor hydrogels treated with and without HAVDI was 0.35 and 0.10, respectively (Figure 2D). Introduction of HAVDI (2 mM) to Stiff hydrogels also resulted in a significantly decreased nuclear YAP ratio, 2.18 to 1.72 and decreased nuclear RUNX2 ratio, 5.76 to 2.62 (Figures 2E-F). There was no significant difference in circularity, cell area, or nuclear YAP and RUNX2 ratios among the cells cultured on soft gels and ones cultured on stiff HAVDI gels (Figures 2C-F).

Conclusions: These morphological changes in cells cultured on a stiff environment including HAVDI suggest that the HAVDI cell-cell mimetic peptides can work to attenuate matrix mechanosensing. The reduction of nuclear YAP and RUNX2 further corroborates this finding and suggests that the downstream effects on protein transcription are also being attenuated. Ongoing work includes a comparison of stemness surface markers (via flow cytometry) between MSCs cultured on tissue culture plastic (loss of stemness over time) and MSCs cultured on softer hydrogels functionalized with HAVDI peptides (maintenance of stemness over time) over multiple passages, allowing for the clinical utilization of MSCs.

Acknowledgements (Optional):  This work is supported by a National Science Foundation (NSF) CAREER award 2239922.