Poster O10 - Enhancing Immunoregulatory Functions of Human Mesenchymal Stromal Cells when Cultured on Peptoid Crosslinked Hydrogel Supplemented with Interferon‐Gamma

Introduction: The stem cell manufacturing field requires high production of relevant potent cells to achieve a therapeutic effect. Supplementation of soluble factors such as Interferon Gamma (IFN-γ) has been crucial in promoting the manufacture of hMSC therapeutic potency by enhancing the secretion of immunoactive factors, but they negatively impact hMSC proliferative capacity. However, soft hydrogels have shown promise for increasing immunomodulatory potential while eliminating IFN-γ’s antiproliferative effect.

Materials and

Methods: In this study, hydrogels were crosslinked with peptoids of different secondary structures to generate substrates of various bulk stiffness but fixed network connectivity. Peptoid crosslinkers with different secondary structures consisted of: 1) a helical, molecularly stiff peptoid, 2) a non-helical, less stiff peptoid, and 3) an unstructured, relatively flexible peptoid (Figure 1A). FTIR was used to confirm the presence of the functional groups of the hydrogel components and the resulting peptoid and peptide-crosslinked hydrogels. SEM was used to visualize the dried structure of each of the hydrogel formulations and the compressive modulus of each hydrogel was analyzed by dynamic mechanical analysis (DMA). To gain a deeper understanding of how substrate stiffness controls hMSC immunomodulatory potential, here we investigated the secretion of seven l human cytokines and chemokines from hMSCs seeded on our hydrogels, including interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), VEGF, epidermal growth factor (EGF), interferon-gamma-induced protein 10 (IP-10), and fibroblast growth factor 2 (FGF-2), which have been shown to play key roles in hMSC immunoregulation. We also evaluated the immunosuppressive properties of hMSCs cultured on peptoid crosslinked hydrogels by assessing suppression of peripheral blood mononuclear cell (PBMC) proliferation during direct contact coculture experiments. To probe intracellular signaling pathways linking matrix mechanics to secretion, we studied the activation of the nuclear factor kappa B (NF-𝜅B) signaling pathway for hMSCs seeded on the developed hydrogels. Finally, we assessed the ability of the hMSCs to differentiate into multiple lineages after culture on our developed substrates.

Results, Conclusions, and Discussions: FTIR analysis confirmed that all spectra strongly display the characteristic peaks of the hyaluronic acid backbone and show clear incorporation of each crosslinker. The dried hydrogel microstructures investigated via SEM showed amorphous morphologies. DMA demonstrated that crosslinker structure modulates the compressive moduli of the resulting hydrogels (Figure 1B). Secretion of interleukin IL-6, MCP-1, MCS-F, and VEGF was shown to depend on hydrogel stiffness in the presence of IFN-γ supplementation, with soft substrates further improving immunoregulation (Figure 1C).
We found significant upregulation of IL-6, MCP-1, and M-CSF that corresponded to suppression of PBMC proliferation on softer hydrogel substrates (unstructured peptoid cross-linker) (Figure 1D). The regenerative potential of the hMSCs was likewise increased on softer substrates, as indicated by the production of VEGF, a marker of wound healing. Immunofluorescent studies demonstrated that IFN-𝛾 and soft hydrogels modulate the immunoregulatory potential of hMSCs through activation of the NF-𝜅B pathway. Our findings further indicate that the ability of hMSCs to differentiate and attach is not altered by the substrate stiffness investigated here. In conclusion, this study highlights the importance of hydrogel stiffness in dictating the immunoregulatory potential of hMSCs in the presence of IFN-𝛾 and serves as a steppingstone toward the development of optimized hydrogel systems that can support hMSC growth and promote the desired cellular properties for therapeutic applications.

Acknowledgements (Optional):