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Tissue Engineering

Tissue Engineering - Poster Session B

Poster L2 - Dynamic Modulation of Hydrogel Stiffness to Enhance Vasculogenesis of Human Induced Pluripotent Stem Cell Derived Endothelial Progenitors

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

Introduction:  Human induced pluripotent stem cells (hiPSCs) hold significant promise for vascular tissue engineering. By differentiating hiPSCs into endothelial progenitors (EPs), these cells can mature into endothelial and smooth muscle cells, the two main cell types found in mature vasculature. hiPSC-derived EPs (hiPSC-EPs) spontaneously form capillary-like structure when encapsulated in 3D hydrogels. The mechanical properties of hydrogels, particularly its stiffness, affects cell behavior during both embryonic development and disease, and have a well-established impact on the degree of vasculogenesis. Since extracellular matrix stiffness changes while vasculogenesis occurs in vivo [1], we hypothesize that by better emulating these processes in vitro, we can enhance vascular network formation. Our research aims to explore how dynamically modulating hydrogel stiffness influences the development of vascular networks. We will employ advanced imaging techniques to monitor cell behavior and vascular formation, providing insights into the mechanotransduction pathways that govern vasculogenesis. This approach seeks to optimize biomaterials for improved vascular tissue engineering applications.

1. Dodson et al. Pediatric Research, 2013

Materials and

Methods:  We differentiated hiPSCs into CD34+-hiPSC-EPs using a modified protocol from Jalilian et al [1]. For cell visualization, hiPSCs were transduced using a lentiviral vector to express tdTomato. CD34+tdTomato+EPs were isolated by fluorescence activated cell sorting and encapsulated in type I Collagen and norbornene-modified hyaluronic acid (Collagen/NorHA) IPN hydrogels as described previously [2, 3]. These hydrogels were characterized by varying crosslinking densities and UV exposure. The cells’ tdTomato expression enables daily monitoring of vascular plexus formation throughout the 7-day culture period using confocal microscopy. Cell polarization and network formation were analyzed using a previously developed computation pipeline [2] and used to determine time points of interest for stiffening the hydrogels. We will stiffen cell-laden hydrogels by swelling in additional crosslinker and photoinitiator followed by exposing the hydrogels to UV light to increase crosslinking density. Hydrogel stiffening was verified using parallel plate rheometry, fluorescence recovery after photobleaching (FRAP), measuring swelling ratios, and degradability. In addition, we will perform FRAP at multiple points throughout the depth of the hydrogel to determine if crosslinking is uniform through the hydrogel depth (in the z-axis direction). We will then stiffen CD34+-hiPSC-EP-laden hydrogels at different time points during vasculogenesis and use our computational pipeline to determine how changes in situ stiffness during different stages of vasculogenesis impact plexus formation.

1. Jalilian et al. Biorxiv, 2020
2. Crosby et al. Tissue Engineering Part A, 2019
3. Crosby et al. Acta Biomaterialia, 2020

Results, Conclusions, and Discussions: We have previously encapsulated CD34+-hiPSC-EPs in Collagen/NorHA IPNs at different crosslinking densities to evaluate how changing stiffness affects vasculogenesis [1]. However, this experiment did not include the full range of possible crosslinking densities and only quantified vasculogenesis at the end of the 7-day culture period. Because we are interested in changing the cells’ extracellular environment during vascular network formation, we utilized a tdTomato-expressing hiPSC line that enables live imaging of the cell-laden hydrogels as the hiPSC-EPs are undergoing vasculogenesis. Figure 1a shows representative z-stacks of CD34+-hiPSC-EP-laden hydrogels at 25% crosslinking, which refers to the percentage of available norbornene groups occupied by crosslinker, at each day of the culture period. We have verified that the cells retain their tdTomato expression for all 7 days and form robust capillary-like vessels with visible lumens. There is also a visible increase in network connectivity starting on day 4 of culture, and we hypothesize that stiffening the hydrogels at this time point will have the greatest effect on vascular network formation. Future work will include repeating this experiment for different crosslinking densities as well as modifying our existing computational pipeline to better track vasculogenesis over time.
In addition, we performed FRAP to verify successful stiffening of Collagen/NorHA IPNs, correlating differences in diffusivity with hydrogel stiffness. We found, as expected, a decrease in the diffusion coefficient between the 25% and 50% crosslinked hydrogels. We also tested a hydrogel stiffened from 25% to 50% using 10 mW/cm2 light for 50 seconds, the same parameters used for initial hydrogel crosslinking, but we found that there was only a statistically significant change in diffusivity near the top of the hydrogel (Figure 1b). Increasing the exposure duration to 120 seconds resulted in a linear change in diffusivity from the top of the hydrogel to the bottom. In the future, we plan on modifying our stiffening protocol to reduce the effects of the stiffness gradient. In addition, we plan on further verifying successful stiffening using the material characterization methods described in the materials and methods section.

1. Crosby et al. Acta Biomaterialia, 2020

Acknowledgements (Optional):  We would like to thank Bryce Larsen for generating the tdTomato-expressing hiPSC line. We gratefully acknowledge the financial support of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), and the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (grants R21EB027812-01A1 and R01HL15829, respectively).