Multifunctional biomimetic hydrogel systems to boost the immunomodulatory potential of mesenchymal stromal cells

Abstract

Mesenchymal stromal cells (MSCs) hold great therapeutic potential, in part because of their immunomodulatory properties. However, these properties can be transient and depend on multiple factors. Here, we developed a multifunctional hydrogel system to synergistically enhance the immunomodulatory properties of MSCs, using a combination of sustained inflammatory licensing and three-dimensional (3D) encapsulation in hydrogels with tunable mechanical properties. The immunomodulatory extracellular matrix hydrogels (iECM) consist of an interpenetrating network of click functionalized-alginate and fibrillar collagen, in which interferon γ (IFN-γ) loaded heparin-coated beads are incorporated. The 3D microenvironment significantly enhanced the expression of a wide panel of pivotal immunomodulatory genes in bone marrow-derived primary human MSCs (hMSCs), compared to two-dimensional (2D) tissue culture. Moreover, the inclusion of IFN-γ loaded heparin-coated beads prolonged the expression of key regulatory genes upregulated upon licensing, including indoleamine 2,3-dioxygenase 1 (IDO1) and galectin-9 (GAL9). At a protein level, iECM hydrogels enhanced the secretion of the licensing responsive factor Gal-9 by hMSCs. Its presence in hydrogel conditioned media confirmed the correct release and diffusion of the factors secreted by hMSCs from the system. Furthermore, co-culture of iECM-encapsulated hMSCs and activated human T cells resulted in suppressed proliferation, demonstrating direct regulation on immune cells. These data highlight the potential of iECM hydrogels to enhance the immunomodulatory properties of hMSCs in cell therapies.

Keywords: Alginate; Extracellular matrix; Hydrogel; Immunomodulation; Interferon; MSCs.

Fig. 1.. Immunomodulatory gene expression by 2D tissue cultured hMSCs on day 3.

(A) Schematic representation of the experimental procedure. After hMSC overnight licensing with IFN-γ and TNF-α, cells were detached and 2D seeded on tissue culture plates (TCP). At different time-points, RNA was isolated from the cells for the subsequent RT-qPCR analysis. (B) Normalized gene expression of IDO1, GAL9, PTGS2, IL1RN, HGF and IL10 by tissue culture hMSCs after overnight licensing with IFN-γ and TNF-α compared to control untreated cells. Normalized to the housekeeping gene GAPDH. * Statistically significant difference between control and licensed hMSCs, Studenťs T test, unpaired. p < 0.05, **p < 0.01. (C) Normalized gene expression of IDO1, GAL9, PTGS2, IL1RN, HGF and IL10 by hMSCs on tissue culture 3 days after overnight licensing with IFN-γ and TNF-α. Normalized to GAPDH and day 0 expression. * Statistically significant difference between day 0 and day 3, Studenťs T test, unpaired for IDO1, GAL9, PTGS2, HGF and IL10. Mann–Whitney U test for IL1RN. ***p < 0.001, **p < 0.01. hMSCs, human mesenchymal stromal cells. IFN-γ, interferon γ. TNF-α, tumor necrosis factor α.

Fig. 2.. aECM hydrogels.

(A) Schematic representation of the experimental procedure. After hMSC overnight preconditioning with IFN-γ and TNF-α, cells were detached and 3D encapsulated in aECM hydrogels. At different time-points, RNA was isolated from the cells for the subsequent RT-qPCR analysis. (B) aECM hydrogel system structure. (C) When alginates were crosslinked with calcium, viscous hydrogels were obtained, (D) whereas the combination of ionic crosslinking and covalent crosslinking between the Norborene and Tetrazine groups led to more elastic gels. aECM, artificial extracellular matrix. Nb, norborene. Tz, tetrazine. hMSCs, human mesenchymal stromal cells.

Fig. 3.. Immunomodulatory gene expression by hMSCs 3D cultured in aECM hydrogels on day 3.

Normalized gene expression of (A) IDO1, (B) GAL9, (C) PTGS2, (D) IL1RN, (E) HGF and (F) IL10 by hMSCs encapsulated within aECM hydrogels 3 days after overnight licensing with IFN-γ and TNF-α and subsequent encapsulation. Normalized to GAPDH. * Statistically significant differences, one-way ANOVA with Tukey's post hoc test for IDO1, GAL9 and IL1RN; one-way ANOVA with Tamhane’s post hoc test for PTGS2, HGF and IL10. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to 2D cultured hMSCs. aECM, artificial extracellular matrix. hMSCs, human mesenchymal stromal cells. IFN-γ, interferon γ. TNF-α, tumor necrosis factor α.

Fig. 4.. Immunomodulatory gene expression by aECM encapsulated hMSCs over time.

Normalized gene expression of (A) IDO1, (B) GAL9, (C) PTGS2, (D) IL1RN, (E) HGF and (F) IL10 by aECM encapsulated hMSCs 0, 3 and 7 days after licensing with IFN-γ and TNF-α and subsequent encapsulation. Normalized to GAPDH. * Statistically significant difference between days 3 and 7, Studenťs T test, unpaired. *p < 0.05, **p < 0.01 and ***p < 0.001. hMSCs, human mesenchymal stromal cells. IFN-γ, interferon γ. TNF-α, tumor necrosis factor α.

Fig. 5.. Characterization of the new iECM materials.

(A) Schematic representation of iECM hydrogel types. (B) Phase microscopy images of hMSCs encapsulated in iECM hydrogels. Scale bar = 200 μm. (C) hMSC viability in aECM and iECM hydrogels on day 7 after overnight licensing with IFN-γ and TNF-α and subsequent encapsulation. Normalized to the total cell count in each hydrogel. n.s.d., no significant differences by one-way ANOVA. aECM, artificial extracellular matrix. iECM, immunomodulatory extracellular matrix. IFN-γ, interferon γ. hMSC, human mesenchymal stromal cells.

Fig. 6.. Immunomodulatory gene expression by hMSCs encapsulated in iECM hydrogels on day 7.

Normalized gene expression of (A) IDO1, (B) GAL9, (C) PTGS2, (D) HGF, (E) IL10 and (F) IL1RN by hMSCs encapsulated within iECM hydrogels 7 days after overnight licensing with IFN-γ and TNF-α and subsequent encapsulation. Normalized to GAPDH. Statistical significance by one-way ANOVA with Tukey's post hoc test for IDO1, PTGS2, HGF, IL10 and IL1RN; one-way ANOVA with Tamhane’s post hoc test for GAL-9. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to aECM. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to blank iECM. aECM, artificial extracellular matrix. iECM, immunomodulatory extracellular matrix. hMSC, human mesenchymal stromal cells.

Fig. 7.. Immunomodulatory effects of hMSCs encapsulated in iECM hydrogels.

(A-B) Gal-9 and (C-D) IL-1Ra protein levels in hydrogel conditioned media. * Statistically significant differences, one-way ANOVA with Tukey's post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001. (E-F) Flow cytometry histogram (E) and mean fluorescence intensity (F) of CFSE-stained T cells co-cultured with IFN-γ or Blank iECM hydrogel encapsulated hMSCs (1:1.25 hMSC:T cell ratio) compared to control without co-culture (Ctrl). iECM: immunomodulatory extracellular matrix. hMSCs: human mesenchymal stromal cells. IFN-γ: interferon γ. Ctrl: control (no hMSC). MFI: mean fluorescence intensity. * Statistically significant differences, Studenťs T test, unpaired. *p < 0.05, **p < 0.01 and ***p < 0.001.