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. 2012 Oct;91(10):948-54.
doi: 10.1177/0022034512458690. Epub 2012 Aug 17.

Fas ligand regulates the immunomodulatory properties of dental pulp stem cells

Y Zhao  1 , L Wang, Y Jin, S Shi
Affiliations

Fas ligand regulates the immunomodulatory properties of dental pulp stem cells

Y Zhao et al. J Dent Res. 2012 Oct.

Abstract

Dental pulp stem cells (DPSCs) possess immunoregulatory properties, but the underlying mechanism is not fully understood. Here we showed that DPSCs were capable of inducing activated T-cell apoptosis in vitro and ameliorating inflammatory-related tissue injuries when systemically infused into a murine colitis model. Mechanistically, DPSC-induced immunoregulation was associated with the expression of Fas ligand (FasL), a transmembrane protein that plays an important role in inducing the Fas apoptotic pathway. Knockdown of FasL expression by siRNA in DPSCs reduced their capacity to induce T-cell apoptosis in vitro and abolished their therapeutic effects in mice with colitis. However, the expression level of FasL did not affect either DPSC proliferation rate or multipotent differentiation potential. In summary, FasL governs the immunoregulatory property of DPSCs in the context of inducing T-cell apoptosis.

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Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.
Figure 1.
Characterization of rDPSCs. (A) rDPSCs generated markedly higher numbers of CFU-F than did rBMMSCs. (B) rDPSCs showed an increased proliferation rate in comparison with that of rBMMSCs, as determined by BrdU incorporation assay. (C) rDPSCs showed more elevated population doublings than did rBMMSCs. (D) Flow cytometric analysis showed that rDPSCs expressed surface molecules CD90, CD29, CD73, and CD106. (E) Immunocytochemistry staining further confirmed that rDPSCs expressed CD90 and CD29. (F) Alizarin Red staining showed that rDPSCs had a lower capacity than rBMMSCs to form mineralized nodules after culture in osteoinductive conditions for 4 wks. Western blot analysis confirmed that rDPSCs expressed elevated levels of the osteogenic genes ALP, Runx2, and OCN in the osteoinductive group. (G) rDPSCs were capable of forming mineralized tissue when transplanted subcutaneously into immunocompromised mice with hydroxyapatite tricalcium phosphate (HA/TCP) as a carrier. rDPSCs formed less mineralized tissue when compared with rBMMSCs. B = bone, M = mineralized tissue, BM = bone marrow, H = HA/TCP carrier. (H) Oil Red O staining showed that rDPSCs had reduced capacity to differentiate into adipocytes compared with rBMMSCs at 2 wks post-adipogenic induction. Western blot analysis confirmed that adipogenic induction up-regulated expression of the adipogenic genes PPARγ-2 and LPL.
Figure 2.
Figure 2.
FasL is required for rDPSC-based immunomodulation. (A) Flow cytometric analysis showed that ~9.63% of cultured rDPSCs at passage 3 co-expressed FasL with CD90. (B) Immunofluorescence confirmed that rDPSCs express FasL. (C) Western blot analysis showed that FasL siRNA could knock down FasL expression in rDPSCs. (D, E) When co-cultured with T-cells, FasL-knockdown rDPSCs showed a reduced capacity to inhibit T-cell viability. Apoptosis assay confirmed that FasL-knockdown rDPSCs showed a reduced capacity to induce 7AAD/Annexin-positive apoptotic T-cells when compared with the control siRNA group. (F) FasL-knockdown rDPSCs showed a reduced capacity to induce iTh17 cell apoptosis compared with the control siRNA group. No significant difference was observed in the induction of iTreg apoptosis between the rDPSC FasL-siRNA group and the rDPSC control siRNA group. (G) Systemic infusion of control rDPSCs (control siRNA) protected mice from colitis-induced body weight loss. (H) HE staining showed the infiltration of inflammatory cells (arrow) in the colon, with destruction of the epithelial layer (triangles) in mice with colitis. rDPSC, but not FasL siRNA rDPSC, transplantation rescued the disease phenotype in the colon and reduced the histological activity index. *p < 0.05. Bar length = 20 µm.
Figure 3.
Figure 3.
Human DPSCs express FasL to induce T-cell apoptosis. (A) Flow cytometric analysis showed that human DPSCs (hDPSCs) expressed CD29, CD73, CD105, and CD90. (B) Flow cytometric analysis showed that cultured hDPSCs co-expressed CD105 with FasL. (C) Western blot analysis showed that siRNA reduced FasL expression in hDPSCs. (D) When co-cultured with T-cells, FasL-knockdown hDPSCs showed a reduced capacity to inhibit T-cell viability. (E) Apoptosis assay confirmed that FasL-knockdown hDPSCs showed a reduced capacity to induce 7AAD/Annexin-positive apoptotic T-cells when compared with the siRNA-negative control group. *p < 0.05. Bar length = 20 µm.
Figure 4.
Figure 4.
FasL expression has no impact on rDPSC proliferation and differentiation. (A) Knockdown FasL expression by siRNA did not affect rDPSC proliferation, as assessed by BrdU labeling assay. (B) FasL-knockdown by siRNA failed to alter the expression of surface molecules CD29, CD90, CD73, CD106, CD49, CD34, CD45, and CD11b in rDPSCs. (C) Western blot analysis confirmed that knockdown FasL expression by siRNA did not change the expression of CD90 in rDPSCs. (D) Osteogenic differentiation capacity of rDPSCs was not altered by FasL knockdown, as indicated by Alizarin Red staining, to show mineralized nodule formation and by Western blot analysis to show expression levels of the osteogenic genes ALP, Runx2, and OCN. (E) In vivo implantation assay showed that FasL knockdown rDPSCs formed an amount of mineralized tissue similar to that observed in control rDPSCs. (F) FasL knockdown was unable to alter adipogenic differentiation capacity, as indicated by the number of Oil Red O–positive cells and expression of the PPARγ2 and LPL.
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