Mesenchymal stem cells derived from human induced pluripotent stem cells modulate T-cell phenotypes in allergic rhinitis

Abstract

Background: Human induced pluripotent stem cells (iPSCs) possess remarkable self-renewal capacity and the potential to differentiate into novel cell types, such as mesenchymal stem cells (MSCs). iPSC-MSCs have been shown to enhance tissue regeneration and attenuate tissue ischaemia; however, their contribution to the immune regulation of Th2-skewed allergic rhinitis (AR) and asthma remains unclear.

Objective: This study compared the immunomodulatory effects of iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on lymphocyte proliferation, T-cell phenotypes and cytokine production in peripheral blood mononuclear cells (PBMCs) in patients with AR, and investigated the possible molecular mechanisms underlying the immunomodulatory properties of iPSC-MSCs.

Methods: In co-cultures of PBMCs with iPSC-MSCs or BM-MSCs, lymphocyte proliferation was evaluated using 3H-thymidine (3H-TdR) uptake, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) assays; the regulatory T-cell (Treg) phenotype was determined by flow cytometry, and cytokine levels were measured using an enzyme-linked immunosorbent assay. The immunomodulatory properties of both MSCs were further evaluated using NS398 and transwell experiments.

Results: Similar to BM-MSCs, we determined that iPSC-MSCs significantly inhibit lymphocyte proliferation and promote Treg response in PBMCs (P < 0.05). Accordingly, the cytokine milieu (IFN-γ, IL-4, IL-5, IL-10 and IL-13) in the supernatants of PBMCs changed significantly (P < 0.05). The immunomodulatory properties of iPSC-MSCs and BM-MSCs were associated with prostaglandin E2 (PGE2) production and cell-cell contact.

Conclusions: These data demonstrate that iPSC-MSCs are capable of modulating T-cell phenotypes towards Th2 suppression through inducing Treg expansion, suggesting that iPSC-MSCs can be used as an alternative candidate to adult MSCs to treat allergic airway diseases.

Figure 1

The immunosuppressive effect of iPSC-MSCs on PHA-stimulated lymphocyte proliferation. iPSC-MSCs significantly inhibited PHA-stimulated lymphocyte proliferation in a number-dependent manner, as determined by 3H-TdR uptake. BM-MSCs were used as a control. PBMCs from healthy volunteers (1 × 10⁵/well) were stimulated by PHA (5 μg/ml) in a 96-well plate for 3 days in the presence or absence of different numbers of iPSC-MSCs or BM-MSCs (1 × 10⁴/well) (n = 10). The data are expressed as the means ± SEM. *P < 0.05; **P < 0.01.

Figure 2

The immunosuppressive effect of iPSC-MSCs on PHA-stimulated T-cell proliferation. iPSC-MSCs suppressed T-cell proliferation in PHA-stimulated PBMCs, as determined by multicolour flow cytometric analysis. CFDA-SE-labelled PBMCs from healthy volunteers (5 × 10⁵ cells/well) were cultured in a 24-well plate for 3 days, and PBMCs were gated for flow cytometric analysis based on CD3⁺ staining (A). Representative results of T-cell proliferation without stimulation (B), PHA (5 μg/ml) stimulation (C), PHA stimulation in the presence of iPSC-MSCs (5 × 10⁴/well) (D) or BM-MSCs (E) are shown. Both iPSC-MSCs and BM-MSCs significantly increased the ratio of G1 T cells but decreased the ratio of G3 and G4 T cells in PHA-stimulated PBMCs (n = 10) (F). The data are expressed as the means ± SEM. G (generation) indicates the number of cell divisions. *P < 0.05; **P < 0.01.

Figure 3

Induced pluripotent stem cells-MSCs enhanced the Treg frequency in Der p1-stimulated PBMCs, as determined by flow cytometry. PBMCs from allergic rhinitis (AR) patients (1 × 10⁶ cells/well) were cultured in a 24-well plate following Der p1 (10 μg/ml) stimulation in the presence or absence of iPSC-MSCs or BM-MSCs (5 × 10⁴/well) for 3 days. Representative flow cytometric results of CD4⁺CD25⁺ cells (A) and Foxp3⁺CD4⁺ cells (C) are shown. Both iPSC-MSCs and BM-MSCs significantly increased the frequency of Foxp3⁺CD4⁺ cells (D), but not CD4⁺CD25⁺ T cells (B), in the CD4⁺ subpopulation after Der p1 stimulation (n = 5 for each group). The data are expressed as the means ± SEM. *P < 0.05.

Figure 4

Induced pluripotent stem cells-MSCs-modulated cytokine levels in the supernatants of Der p1-stimulated PBMCs. PBMCs (1 × 10⁶ cells/well) collected from patients with AR were cultured in a 24-well plate following Der p1 (10 μg/ml) stimulation in the presence or absence of iPSC-MSCs or BM-MSCs (5 × 10⁴/well) for 3 days. The levels of IFN-γ, IL-4, IL-5, IL-13 and IL-10 in the supernatants of Der p1-stimulated PBMCs were determined using ELISA. iPSC-MSCs significantly increased the levels of IFN-γ (A) and IL-10 (B) but decreased the levels of IL-4 (C), IL-5 (D) and IL-13 (E) in the supernatants of Der p1-stimulated PBMCs. For each group, n = 11. The data are expressed as the means ± SEM. *P < 0.05; **P < 0.01. nd: no detectable.

Figure 5

Induced pluripotent stem cells-MSCs or BM-MSCs modulated the levels of TGF-β1 and PGE2 in the supernatants of PHA or Der p1-stimulated PBMCs. (A, C) PBMCs from healthy donors (1 × 10⁵ cells/well) were cultured in a 96-well plate in the presence of iPSC-MSCs or BM-MSCs (1 × 10⁴ cells/well) for 3 days under the indicated conditions. (B, D) PBMCs from patients with AR (1 × 10⁶ cells/well) were cultured in a 24-well plate with 10 μg/ml Der p1 in the presence of iPSC-MSCs or BM-MSCs (5 × 10⁴ cells/well) for 3 days. Serum-free medium was used for the TGF-β1 experiments. The levels of TGF-β1 and PGE2 in the supernatants were determined using ELISA. A significant effect of both iPSC-MSCs and BM-MSCs on TGF-β1 level was not observed in the supernatants of PHA- or Der p1-stimulated PBMCs (A, B). However, both iPSC-MSCs and BM-MSCs significantly increased the PGE2 level in the supernatants of PHA- or Derp1-stimulated PBMCs, which was significantly inhibited by NS398 (C, D). For each group, n = 6. The data are expressed as the means ± SEM. **P < 0.01. nd: no detectable.

Figure 6

The immunomodulatory effects of iPSC-MSCs or BM-MSCs on lymphocyte proliferation and T regulatory cells' association with PGE2 production and cell–cell contact suppression. PBMCs from healthy volunteers were stimulated by PHA (5 μg/ml) for 3 days in the presence of iPSC-MSCs or BM-MSCs with NS398 (5 μM) or with transwell separation from MSCs. NS398 (A) and transwell separation (B) significantly reversed the immunomodulatory effect of iPSC-MSCs and BM-MSCs on PHA-stimulated lymphocyte proliferation, as suggested by 3H-TdR uptake (n = 10). PBMCs (1 × 10⁶ cells/well) collected from patients with AR were cultured in a 24-well plate with Der p1 (10 μg/ml) stimulation in the presence of iPSC-MSCs or BM-MSCs (5 × 10⁴ cells/well) with NS398 or without transwell separation from MSCs for 3 days. The transwell experiment significantly reversed the immunomodulatory effects of iPSC-MSC and BM-MSCs on the frequency of Foxp3⁺CD4⁺ cells in the CD4⁺ subpopulation of cultured PBMCs (C). Representative flow cytometric results in groups (D, Der p1-stimulated PBMCs; E, Der p1-stimulated PBMCs plus iPSC-MSCs; F, Der p1-stimulated PBMCs plus BM-MSCs) in PBMCs are shown. For each group, n = 5. The data are expressed as the means ± SEM. *P < 0.05, **P < 0.01. nd: no detectable.