Hypoxia, a dynamic tool to amplify the gingival mesenchymal stem cells potential for neurotrophic factor secretion

Abstract

Gingival mesenchymal stem cells (GMSCs) have significant regenerative potential. Their potential applications range from the treatment of inflammatory diseases, wound healing, and oral disorders. Preconditioning these stem cells can optimize their biological properties. Hypoxia preconditioning of MSCs improves stem cell properties like proliferation, survival, and differentiation potential. This research explored the possible impact of hypoxia on the pluripotent stem cell properties that GMSCs possess. We evaluated the morphology, stemness, neurotrophic factors, and stemness-related genes. We compared the protein levels of secreted neurotrophic factors between normoxic and hypoxic GMSC-conditioned media (GMSC-CM). Results revealed that hypoxic cultured GMSC's had augmented expression of neurotrophic factors BDNF, GDNF, VEGF, and IGF1 and stemness-related gene NANOG. Hypoxic GMSCs showed decreased expression of the OCT4 gene. In hypoxic GMSC-CM, the neurotrophic factors secretions were significantly higher than normoxic GMSC-CM. Our data demonstrate that culturing of GMSCs in hypoxia enhances the secretion of neurotrophic factors that can lead to neuronal lineage differentiation.

Keywords: Gingival mesenchymal stem cell; Hypoxia; Neurotrophic factors; Normoxia.

Fig. 1

Isolation and characterization of GMSCs. (A) Photomicrograph of passage 2 with GMSCs. Scale bar = 100 μm. (B-H) Characterization of GMSCs for MSC-specific positive markers CD73, CD90, CD105 and negative markers CD34, CD45, HLA-DR. (GMSCs: Gingival mesenchymal stem cells).

Fig. 2

Comparative characterization of GMSCs incubated at normoxia (∼20% oxygen) and hypoxia (0.5% – 1% oxygen) for MSC-specific markers. (A,B) Photomicrograph of GMSCs after normoxic incubation at day 0 and day 7. (C,D) Photomicrograph of GMSCs after hypoxic incubation at day 0 and day 7. Scale bar = 100 μm. (E-R) Comparative characterization of GMSCs incubated at normoxia and hypoxia for MSC-specific positive markers CD73, CD90, CD105 and negative markers CD34, CD45, HLA-DR. (ns: insignificant, **p < .01).

Fig. 3

RT-qPCR analysis for gene expression in GMSCs incubated at normoxia and hypoxia. (A) Comparative gene expression (fold change) for gene expressing hypoxia-inducible factor (HIF-1α). (B-E) Comparative gene expression (fold change) for genes expressing neurotrophic factors BDNF, GDNF, VEGF, and IGF. (ns: not significant, **p < .01).

Fig. 4

Western blot analysis for protein expression in GMSCs incubated at normoxia and hypoxia. (A, B) Comparative protein expression (optical density) for hypoxia-inducible factor (HIF-1α). (A, C-F) Comparative protein expression (optical density) for neurotrophic factors BDNF, GDNF, VEGF, and IGF. (**p < .01).

Fig. 5

Bead-based flow cytometry analysis of secretome in GMSC-conditioned medium for secreted proteins by GMSCs incubated at normoxia and hypoxia. Comparative protein levels (pg/mL) for neurotrophic factors (A) BDNF, (B) GDNF, (C) VEGF, and (D) IGF. (*p < .05, **p < .01).

Fig. 6

RT-qPCR analysis for gene expression in GMSCs incubated at normoxia and hypoxia. (A, B) Comparative gene expression (fold change) for genes expressing stemness-related transcription factors NANOG and OCT4. (*p < .05, **p < .01).