(C-85) Effects Of Stiffness and Cell-Cell Mimetic Peptides on Stemness and Matrix Mechanosensing

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row C - Poster # 85

Presenting Author(s)

TT

Tyler Torres

Undergraduate Researcher
Rowan University
Swedesboro, New Jersey, United States

Primary Investigator(s)

SV

Sebastian Vega

Assistant Professor
Rowan University, United States

Co-Author(s)

MR

Matthias Recktenwald, MS

Graduate Research Assistant
Rowan University
Philadelphia, Pennsylvania, United States

Introduction:: The authors believe that this abstract best fits the Biomanufacturing Track and Biomaterials for Cell Manufacturing and Tissue Biofabrication Sub-Track. Mesenchymal Stem Cells (MSCs) hold immense potential for cell therapies due to their ability to proliferate and differentiate into musculoskeletal tissue cells including osteoblasts, adipocytes, and chondrocytes. However, their potential for clinical translation is challenged by the inability to expand MSCs in vitro while maintaining their stemness throughout multiple passages. There is growing consensus that this is due to expanding MSCs on tissue culture plastic, resulting in supraphysiological matrix mechanosensing and a progressive loss in stemness. N-cadherin mediated cell-cell interactions attenuate matrix mechanosensing, and the hypothesis of this work is that MSCs cultured on biomaterials functionalized with N-cadherin mimetic peptides will exhibit a decrease in matrix mechanosensing resulting in maintaining their stemness over multiple passages. To test this hypothesis, soft and stiff norbornene-modified hyaluronic acid hydrogels (HANor) were formed with N-cadherin mimetic peptides (HAVDI) and used to study how stiffness and the HAVDI peptide influence MSC morphology and the expression of stemness surface markers over time.

Materials and Methods:: Bone marrow-derived mesenchymal stem cells (MSCs, Lonza, P3 to P4) were cultured in MSC growth medium (α Minimum Essential Medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum). To form hydrogels, norbornene-modified hyaluronic acid (HANor) macromers (2 wt%) were dissolved in PBS in the presence of photoinitiator (Irgacure 2959, 0.05 wt%) and low (0.5 mM) or high (5 mM) crosslinker (dithiothreitol, DTT) concentration to form soft or stiff hydrogels, respectively. Soft and Stiff HANor hydrogels were also formed with thiolated RGD peptide (2 mM) for adhesion, and with or without HAVDI peptide (2 mM) for cell-cell signaling. Solutions were pipetted into cylindrical silicone molds (8 mm diameter, 0.5 mm height) and photopolymerized with ultraviolet light (10 mW/cm², 10 minutes). Hydrogels were incubated in a MSC growth medium for 4 hours to ensure adequate swelling, and MSCs (5,000 cells/cm2) were seeded atop. 24 hours post-plating, samples were fixed with a 10% formalin solution, MSCs permeabilized with a 0.1% Triton X-100 solution, blocked with a 3% BSA (bovine serum albumin) solution. Actin filaments and nuclei were stained using AF568 phalloidin (1:250) and Hoechst (1:1000) solutions, respectively. Samples were imaged with a confocal microscope, and single-cell morphology analysis (area, circularity) was performed using ImageJ software.

Results, Conclusions, and Discussions::

Unconfined compression of cylindrical HANor hydrogels (8 mm diameter, 0.5 mm height) showed that hydrogels formed with low (0.5 mM) and high (5 mM) DTT had Soft and Stiff compressive modulus values of 2.52 ± 0.97 kPa and 15.35 ± 3.40 kPa, respectively (Figure 1A). The average cell area for Soft HANor with and without HAVDI (2 mM) is 370 μm² and 1,330 μm², respectively. The average cell area for Stiff HANor hydrogels treated with and without HAVDI is 1,380 μm² and 3,630 μm², respectively (Figure 1B). Cell circularity was measured on a 0-1 scale with 0 representing a linear line and 1 representing a circle. The average cell circularity for Soft HANor hydrogels treated with and without HAVDI is 0.73 (round) and 0.21 (spread), respectively, while for Stiff hydrogels, it is 0.46 and 0.16, respectively (Figure 1C). Representative images of single MSCs on Soft (Figure 1D) and Stiff (Figure 1E) hydrogels with and without the HAVDI peptide corroborate these findings. Cell spreading can be attributed to matrix mechanosensing, and a decrease in cell spreading in HAVDI peptide-containing groups suggests that cell-cell signals attenuate matrix mechanosensing on Soft and Stiff hydrogels.

In a pilot study, we extracted MSCs from human bone marrow using Ficoll centrifugation and cultured MSCs directly onto 25 kPa and 50kPa hydrogels and TCP as a control (Figure 1F). We found that MSCs attach and grow on softer substrates and flow cytometry of stemness surface marker CD90 showed that a large fraction of MSCs retained their stemness for at least 5 passages on 25 kPa hydrogels, which are orders of magnitude softer than TCP (~3 GPa) (Figure 1G). In ongoing studies, we are interested in studying the combined effects of stiffness and HAVDI peptide dosing on maintaining stemness over multiple passages towards developing novel materials to produce large populations of high quality MSCs. These exciting results have the potential for improving in vitro MSC expansion which would address limitations of current cell manufacturing platforms and thereby increase the therapeutic use of MSCs.

Acknowledgements (Optional): :

This work was supported by the National Science Foundation (NSF) CAREER award 2239922.

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