Implication of NOD1 and NOD2 for the differentiation of multipotent mesenchymal stem cells derived from human umbilical cord blood

Abstract

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are known to trigger an innate immune response against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs), little is known about the role of NLRs on the MSC function. In this study, we investigated whether NOD1 and NOD2 regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs). The genes of TLR2, TLR4, NOD1, and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist (Pam(3)CSK(4) for TLR2, LPS for TLR4, Tri-DAP for NOD1, and MDP for NOD2) led to IL-8 production in hUCB-MSC, suggesting the expressed receptors are functional in hUCB-MSC. CCK-8 assay revealed that none of agonist influenced proliferation of hUCB-MSCs. We next examined whether TLR and NLR agonists affect osteogenic-, adipogenic-, and chondrogenic differentiation of hUCB-MSCs. Pam(3)CSK(4) and Tri-DAP strongly enhanced osteogenic differentiation and ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly did. Treatment of U0126 (MEK1/2 inhibitor) restored osteogenic differentiation enhanced by Pam(3)CSK(4). Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs, but Pam(3)CSK(4) and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs with difference in the ability. Our findings suggest that NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs.

Figure 1. TLRs and NLRs were functionally expressed in hUCB-MSCs.

mRNA expressions of TLR2, TLR4, NOD1, NOD2, and Rip2 were determined by RT-PCR in hUCB-MSCs (A). The cells were treated with Pam₃CSK₄, LPS (B), Tri-DAP, and MDP (C) in a dose-dependent manner for 24 h and IL-8 production was determined using a commercial ELISA kit (B and C).

Figure 2. Activation of TLRs and NLRs did not influence the proliferation of hUCB-MSCs.

hUCB-MSCs were treated with various doses of Pam₃CSK₄, LPS (A), Tri-DAP, and MDP (B) for 4 days and cell proliferation was determined by CCK-8 kit.

Figure 3. Stimulation with TLR and NLR agonists promoted osteogenic differentiation of hUCB-MSCs through phosphorylation or ERK1/2.

hUCB-MSCs were grown in conditioned media at the absence or presence of Pam₃CSK₄, LPS, Tri-DAP, and MDP (10 µg/ml) for 2 weeks, and culture media was replaced twice per week. Osteogenesis was determined by Alizarin Red S at 2 weeks after treatment (A) and optical density was determined using ELISA at 570 nm (B). hUCB-MSCs were treated with Pam₃CSK₄, LPS, Tri-DAP, and MDP for 15, 30, and 60 min and ERK phosphorylation was determined by Western Blot analysis with an anti-phospho-ERK antibody (C). hUCB-MSCs were co-treated with Pam₃CSK₄ and U0126 for 2 weeks and determined by Alizarin Red staining (D) and quantified using ELISA at 570 nm (E). * P<0.05, ** P<0.01, *** P<0.001.

Figure 4. Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs.

hUCB-MSCs were grown in conditioned media at the absence or presence of Pam₃CSK₄, LPS, Tri-DAP, and MDP (10 µg/ml) for 3 weeks, and culture media was replaced twice per week. Adipogenesis was determined by Oil Red O staining (A) and level of intercellular lipid was determined using ELISA at 500 nm at 3 weeks after treatment (B and C). hUCB-MSCs (#1114) from another umbilical cord blood were grown in conditioned media at the absence or presence of each ligands for 3 weeks, and adipogenesis was determined (D) and quantified using ELISA at 500 nm (E). ** P<0.01, *** P<0.001.

Figure 5. Activation of TLRs and NLRs promoted chondrogenic differentiation of hUCB-MSCs.

hUCB-MSCs were prepared as pellets and they were cultured in chondrogenic medium supplemented with 500 ng/ml BMP-2 for 3 weeks. Then, the volume of pellets was measured (A) and stained with toluidine blue (B). * P<0.05.