Development of an iPSC-derived tissue-resident macrophage-based platform for the in vitro immunocompatibility assessment of human tissue engineered matrices

Abstract

Upon implanting tissue-engineered heart valves (TEHVs), blood-derived macrophages are believed to orchestrate the remodeling process. They initiate the immune response and mediate the remodeling of the TEHV, essential for the valve's functionality. The exact role of another macrophage type, the tissue-resident macrophages (TRMs), has not been yet elucidated even though they maintain the homeostasis of native tissues. Here, we characterized the response of hTRM-like cells in contact with a human tissue engineered matrix (hTEM). HTEMs comprised intracellular peptides with potentially immunogenic properties in their ECM proteome. Human iPSC-derived macrophages (iMφs) could represent hTRM-like cells in vitro and circumvent the scarcity of human donor material. iMφs were derived and after stimulation they demonstrated polarization towards non-/inflammatory states. Next, they responded with increased IL-6/IL-1β secretion in separate 3/7-day cultures with longer production-time-hTEMs. We demonstrated that iMφs are a potential model for TRM-like cells for the assessment of hTEM immunocompatibility. They adopt distinct pro- and anti-inflammatory phenotypes, and both IL-6 and IL-1β secretion depends on hTEM composition. IL-6 provided the highest sensitivity to measure iMφs pro-inflammatory response. This platform could facilitate the in vitro immunocompatibility assessment of hTEMs and thereby showcase a potential way to achieve safer clinical translation of TEHVs.

Keywords: Extracellular matrix; Induced pluripotent stem cells; Mass spectroscopy; Resident tissue macrophages; Tissue culture; iPSC-derived macrophages.

Figure 1

Formation of iMϕ precursor factories and characterization of differentiated iMφs. (A) Progression of iMφ precursor factory formation towards the yolk sac-like structures (scale bar indicates 200 μm). (B) Brightfield images of (i) iMϕ precursor showing initial non-adherence and (ii) iMφs after differentiation in DM medium for 7 days, which adhered to the bottom of the well. (C) Flow cytometry assessment of iMφs after differentiation in DM medium for 7 days (n = 3; 50,000 cell for each experiment).

Figure 2

Polarization of iMφs towards pro- and anti-inflammatory states for 72 h. (A) Gene expression in iMφs polarized with M1 stimulation (LPS/IFN-γ) for 72 h. (B) Polarization of iMφs with M2 stimulation (IL-4/IL-13) for 72 h. (C) ELISA raw quantification of iMϕ M0 secreted interleukins after differentiation for 7 days in DM medium (dilution of 1:5000, n = 3) (D–G) ELISA quantification of iMφs secreted interleukins (IL-8, TNFa, IL-1β, IL-6) after conditioning with either no stimuli (M0), pro- (M1) or anti-(M2) inflammatory stimuli. Data is normalized on the iMϕ M0 value. (Tukey’s multiple comparison test *p-value < 0.05, ****p-value < 0.0001).

Figure 3

Polarization of iMφs towards pro- and anti-inflammatory states for 72 h. (A) Flow cytometry analysis of surface markers to assess the polarization of iMφs after M1 or M2 stimulation for 72 h, in comparison to non-polarized M0 cells (n = 3). (B) Flow cytometry density plots of non-polarized and polarized iMφs (M1 and M2 conditions) for the markers CD206 (anti-inflammatory) and CD86 (pro-inflammatory), (2way ANOVA Tukey’s multiple comparisons test, *p-value < 0.05, ***p < 0.0005).

Figure 4

Characterization of potentially immunogenic components within hTEMs. (A) Label-free quantification of the 20 most abundant intracellular proteins expressed as normalized MS1 intensities of at 2W, 4W and 6W-hTEMs samples (n = 4/timepoint). (B) label-free quantification and fold change of the MS1 values between the initial 2W and final 6W-hTEM culture time points. (C) Label-free quantification of DAMPs identified in the matrices, expressed as normalized MS1 intensities at 2W, 4W and 6W-hTEM samples (n = 4/timepoint). (D) label-free quantification and fold change of the MS1 values between the initial 2W and final 6W-hTEM production time points (n = 4/timepoint, t-test with pooled variants, *adj. p-value < 0.05).

Figure 5

Assessment of hTEM immunogenicity. Scaffold along (PGA/P4HB) and bovine fibrin gel-coated PGA/P4HB (PGA/FIB) controls, and hTEMs produced at 2W, 4W, and 6W (n = 3/timepoint), were cultured with iMφs (M0) for 3 and 7 days (n = 3/timepoint). (A) Flow cytometry analysis (n = 3/time point) showed no significant surface marker difference compared to iMφs (M0). (B–E) ELISA quantification of secreted cytokines in supernatants (n = 3/timepoint, IL-6 statistics to 6W-hTEM at 3 days. 2way ANOVA Tukey’s multiple comparisons test *p-value < 0.05, **p < 0.005).