Expression of Functional Toll-Like Receptor 2 on Mouse Embryonic Stem Cells

Abstract

Embryonic stem (ES) cells have the capacity to produce all cell types of the body. Understanding murine ES (mES) cell proliferation, survival, differentiation, and self renewal will enhance knowledge of developmental biology and essential use of ES cells. Recently, Toll Like Receptor (TLR) activation has been shown by others to play a role in influencing the differentiation of hematopoietic stem cells. Previous studies have also shown that TLR activation prevents mesenchymal stem cell differentiation into adipocytes, chondrocytes, and osteocytes and plays a role in bone repair. We hypothesized that certain TLR's would be expressed on mES cells and that the ligands for these expressed TLR's would induce functional activity in the mESC's. Therefore, we wanted to determine if TLRs are expressed on mES cells and if so, are they functional. Three different mES cell lines (R1, CGR8, and E14) were used to determine if TLRs are expressed at the mRNA level using primers for murine TLR1-9 mRNA. We found that TLR's 1, 2, 3, 6, and 9 were expressed at the mRNA level, but TLR's 4, 5, 7, and 8 were not. Based on the availability of antibodies to TLR's, and using flow cytometry, we found expression of TLR2 but not TLR 4 on the surface of all three mES cell lines. TLR ligands were used to treat mES cells in the presence of leukemia inhibitory factor (LIF) for an hour. Activation of TLR2 by its ligand Pam3Cys, a synthetic tri-acyl lipoprotein, on mES cells induced NF-κβ nuclear translocation when compared to ES cells not stimulated with TLR ligands. LPS, the ligand for TLR4 did not induce NF-κβ nuclear translocation on ES cells, consistent with lack of cell surface expression of TLR4 on mES cells. TLR expression and TLR ligand interaction were not associated with changes in the morphology of the mES cells or expression of Oct-4, SSEA-1, KLF-4, or Sox-2, markers for maintenance of the undifferentiated state of mES cells. This suggests that the cells remain in an undifferentiated state even after TLR activation by Pam3Cys in the presence of LIF. Thus our study has identified functionally active TLR2 on the surface of mES cells, information that may be of use to further defining a role for TLR's on ES cells, and for manipulation of other ES cell functions.