L16 - Multicellularity, Culture Duration, and Hydrogel Stiffness Guide Induced Pluripotent Stem Cell-Derived Endothelial Progenitor Contractile Behavior and Hydrogel Remodeling

Introduction: : Human induced pluripotent stem cell (hiPSC) technologies are important developments in regenerative medicine, not only because they hold the potential to produce a variety of tissue types, but also because hiPSCs can be replacements that are engineered to avoid the need for patient immune suppression and can be produced in large numbers. One of the largest hurdles that has yet to be overcome for hiPSC therapy is the lack of vascularization that occurs within hiPSC-derived tissues. Therefore, there is a strong need for understanding of the process of vasculogenesis in hiPSC-derived cells. We and others have developed techniques for differentiating hiPSCs into endothelial progenitors (EPs), [1,2]. Additionally, encapsulation and culturing of hiPSC-EPs in hyaluronic acid (HA)-based hydrogels promotes hiPSC-EP self-assembly into interconnecting lumenized structures resembling capillaries [3,4]. We have seen in norbornene-functionalized HA (NorHA) hydrogels crosslinked with enzyme degradable peptides (EDPs) that hydrogel stiffness significantly affects measures of vasculogenesis such as connectivity and CD31 status [5]. Our current work focuses on developing a detailed understanding of the mechanodynamics of hiPSC-EPs in NorHA hydrogels during the initial stages of vasculogenesis. We hypothesized that before network formation, multi-cell cooperativity would increase hiPSC-EP basal contractile ability and hydrogel remodeling, which would mature over time and would be sensitive to hydrogel stiffness.

Materials and

Methods: : hiPSCs that stably express tdTomato were differentiated into EPs and embedded in NorHA hydrogels containing fluorescent Dragon Green fiducial markers and cross-linked with EDPs of different concentrations to produce varying stiffnesses. Hoechst-stained hiPSC-EPs and the surrounding hydrogel fiducial markers were imaged either 4 or 7 days after gelation on a Nikon AXR confocal microscope fitted with a heated stage and piezo controller. 3D images were taken of single cells or small multi-cell clusters in the basal and fully relaxed cytochalasin-D-treated steady states. With added filtering steps to avoid erroneous bead tracking, the FM-TRACK software [6] was implemented to track the movements of the cell and hydrogel from the fully relaxed state to the basal state so that basal contractility dynamics could be assessed. Furthermore, new software was written in Python to analyze kinematic parameters of the hydrogel.

Results, Conclusions, and Discussions:: In our analysis we first found that hydrogel stiffness, culture maturity, and cellularity significantly affected basal contractile displacement magnitude. We next observed that NorHA gels exhibited significant compressibility. This important observation required modifications of the kinematic analysis to separate the strain measures into volume-changing and deviatoric components. Using these modified methods, we found that increasing both the deviatoric and volumetric strain indices significantly increased when there were decreases in hydrogel stiffness, increases in culture time, and changes from single to multicell groups. Furthermore, multicellularity has synergistic effects on the deviatoric and volumetric strain indices by day 7, indicating that hydrogel compressibility is highly influenced by cell cooperativity as cultures mature. Although volumetric strain index changes were less than the deviatoric strain index, the volumetric strain index is not negligible, especially near cell boundaries. Decomposition of deviatoric and volumetric indices is therefore pivotal for future investigations of the mechanical, kinematic, and material properties of neovascularization systems where compressibility of the matrix is modified. Our future work is aimed at quantifying these differences to allow for an accurate calculation of traction forces and will provide deeper insight of the biomechanical processes by which hiPSC-EP produce neovascularization.

Acknowledgements and/or References (Optional):: Imaging performed at the Center for Biomedical Research Support Microscopy and Flow Cytometry Facility at the University of Texas at Austin (RRID: SCR_021756).
TMW is an NIH F32 post-doctoral fellow (1F32HL167570). This work is funded by the following grant awards: NIH-R01EB032533, NIH-R01HL157829, and UTAUS-FA00000461.
[1] Stern et. al. 2025, Reg Eng & Trans Med
[2] Nguyen et. al. 2021, Cell Stem Cell
[3] Crosby et. al. 2019, Reg Biomats
[4] Crosby et. al. 2021, Acta Biomaterialia
[5] Han et. al. 2025, BioRxiv
[6] Lejeune et. al. 2020, SoftwareX