Retinol/inflammation affect stemness and differentiation potential of gingival stem/progenitor cells via Wnt/β-catenin

Abstract

Background and objective: Inflammatory cytokines impact the course of periodontal disease, repair, and regeneration. Vitamin A and its metabolites are inflammation-modulatory biomolecules, affecting cellular pluripotency. The aim of this study was to investigate the effect of retinol and periodontal inflammatory cytokines (IL-1β/TNF-α/IFN-γ) on pluripotency and proliferative properties of gingival mesenchymal stem/progenitor cells (G-MSCs), for the first time.

Material and methods: Human G-MSCs (n = 5) were STRO-1 immuno-magnetically sorted, characterized and expanded in basic medium (control group), in basic medium with IL-1β (1 ng/mL), TNF-α (10 ng/mL), and IFN-γ (100 ng/mL) (inflammatory group), in basic medium with retinol (20 μmol/L) (retinol group) and with retinol added to the inflammatory group (inflammatory/retinol group). β-catenin levels at 1 hour, cellular proliferation over 14 days, and colony-forming units (CFUs) at 14 days were investigated. Pluripotency gene expressions were examined at 1, 3, and 5 days via reverse transcription-polymerase chain reaction (RT-PCR). Multilineage differentiation potential was evaluated, following 5 days priming, using qualitative and quantitative histochemistry and RT-PCR.

Results: G-MSCs were CD14^- , CD34^- , CD45^- , CD73⁺ , CD90⁺ , CD105⁺ , and showed mesenchymal stem/progenitor cells' hallmarks, CFUs, and multilineage differentiation potential. Intracellular β-catenin significantly declined in the stimulated groups (P < 0.001, Friedman test). Cellular proliferation at 72 hours was most prominent in the control and inflammatory group [Median cell numbers (Q25/Q75); 6806 (4983/7312) and 5414 (4457/7230), respectively], followed by an upsurge in the retinol group. At 14 days, the retinol group exhibited the highest CFUs [Median CFUs (Q25/Q75); 35 (20/58), P = 0.043, Wilcoxon signed-rank]. Nanog was most expressed in the inflammatory and retinol group [Median gene expression/PGK1 (Q25/Q75); 0.0006 (0.0002/0.0014) and 0.0005 (0.0003/0.0008)]. Inflammation significantly upregulated Sox2 expression [0.0002 (0.0008/0.0005)], while its expression was diminished in the retinol and inflammatory/retinol group (P < 0.001, Friedman test). Inflammatory/retinol group exhibited the highest multilineage differentiation potential.

Conclusion: Controlled short-term inflammatory/retinol stimuli activate the Wnt/β-catenin pathway, affecting G-MSCs' pluripotency, proliferation, and differentiation. The present findings provide further insights into the inflammatory-regenerative interactions and their modulation potential for G-MSCs-mediated periodontal repair/regeneration.

Keywords: inflammation; interferons; interleukin-1; stem cells; tumor necrosis factor-alpha; vitamin A.