TlR expression profile of human gingival margin-derived stem progenitor cells

Abstract

Background: Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile.

Material and methods: Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs' incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated.

Results: G-MSCs showed all stem/progenitor cells' characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test).

Conclusions: The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions.

Figure 1

Microscopic appearance, colony-formation, surface marker expression and differentiation potential: (A) Phase contrast microscopic appearance of the adherent tissue mass with outgrowing cells (2nd week). (B) Colony-forming units of G-MSCs (crystal violet). (C) Flow cytometric analysis of the surface marker expression profile of G-MSCs. Multilineage differentiation potential of G-MSCs. Alizarin Red staining of the osteogenically stimulated G-MSCs (D) and their controls (E) Oil-Red-O staining of the adipogenically stimulated G-MSCs (F) and their controls (G). Alcian Blue staining of the chondrogenically stimulated G-MSCs (H) and their controls (I).

Figure 2

Median Flouresence Intensity (MFI) of expressed TLRs in G-MSC and G-MSC-i: (A) Median Fluorescence Intensity (MFI) of expressed TLRs 1-10 of G-MSCs (green curve) and of their isotype controls (red curve) after incubation in basic medium. (B) Protein expression of TLRs 1-10 (n=5;box- and whisker plots with medians and quartiles). (C) Median Fluorescence Intensity (MFI) of expressed TLRs 1-10 of G-MSCs (green curve) and of their isotype controls (red curve) after incubation in inflammatory medium (G-MSCs-i). (D) Protein expression of TLRs 1-10 after incubation in inflammatory medium (n=5; box- and whisker plots with medians and quartiles). The green colored boxes show increased MFI after stimulation by the inflammatory medium (Wilcoxon Signed Rank test, statistical significance marked with asterisk, *:p<0.05).

Figure 3

Differences in mRNA expression of TLRs 1-10 in G-MSCs and G-MSC-i: (A) m-RNA expression of TLRs 1-10 in uninflamed condition. (B) m-RNA expression of TLRs 1 to 10 in inflamed condition (n=5; box- and whisker plots with medians and quartiles). The green colored boxes show increased mRNA expression after stimulation by the inflammatory medium (Wilcoxon Signed Rank test, statistical significance marked with asterisk, *:p<0.05).