The effects of hypoxia on the stemness properties of human dental pulp stem cells (DPSCs)

Abstract

Recent studies have demonstrated that culture under hypoxia has beneficial effects on mesenchymal stem cells (MSCs). However, there are limitations to achieving a stable condition in conventional hypoxic CO₂ incubators. DPSCs are a unique type of MSCs which are promising in many regenerative therapies. In this study, we investigated the ideal hypoxic culture environment for DPSCs using a new system that can provide controlled O₂ environment. The effects of hypoxia (3%, 5%) on the stemness properties of DPSCs. Their morphology, proliferation rate, expression of stem cell markers, migration ability, mRNA expression of angiogenic/neurotrophic factors and immunomodulatory genes were evaluated and compared. Additionally, the effect of the discrete secretome on proliferation, migration, and neurogenic induction was assessed. Hypoxic DPSCs were found to be smaller in size and exhibited larger nuclei. 5% O₂ significantly increased the proliferation rate, migration ability, expression of stem cell markers (CXCR4 and G-CSFR), and expression of SOX2, VEGF, NGF, and BDNF genes of DPSCs. Moreover, secretome collected from 5%O₂ cultures displayed higher stimulatory effects on proliferation and migration of NIH3T3 cells and on neuronal differentiation of SH-SY5Y cells. These results demonstrate that 5%O₂ may be ideal for enhancing DPSCs growth, stem cell properties, and secretome trophic effect.

Figure 1. DPSCs exhibited better morphology under hypoxic conditions.

(a) Representative phase contrast micrographs of DPSCs (5^th) cultured in different oxygen tension. (b) Representative micrographs of DPSCs (5^th) cultured in different oxygen tensions stained with Giemsa stain. (c) Cell size as analyzed by Image J. (d) Nucleus size as analyzed by Image J. (mean ± S.E., n = 4. *p < 0.05, ***p < 0.001).

Figure 2. 5% oxygen cultures exhibited higher stem cell surface markers expression and migration ability.

(a) Expression of surface stem cell markers. (b) Migrating cell number. (mean ± S.E., n = 4. *p < 0.05, **p < 0.01).

Figure 3. Hypoxic cultures demonstrated better proliferation rate, higher expression of pluripotency markers, angiogenic/neurotrophic factors, and immunomodulatory genes.

(a) Cell count. (b) Quantitative RT-PCR for mRNA levels of pluripotency markers and angiogenic/neurotrophic factors. (c) Quantitative RT-PCR for mRNA levels of immunomodulatory genes (mean ± S.E., n = 4. *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4. Secretome collected from 5% hypoxic cultures showed better effect on proliferation activity, migration ability, and neurite outgrowth.

(a) Proliferation activity(*p < 0.05, ***p < 0.001) and (b) The migratory activity of NIH3T3 cells supplemented with secretome of each oxygen culture, (mean ± S.E., n = 4) 5% vs. 20% (*p < 0.05, **p < 0.01, ***p < 0.001), 3% vs. 20% (^#p < 0.05, ^##p < 0.01, ^###p < 0.001), 5% vs. 3% (^§p < 0.05, ^§§p < 0.01) (c) Representative photos demonstrating the morphology of SH-SY5Y supplemented with secretome of each oxygen culture. (d) Quantitative analysis of SH-SY5Y neurite outgrowth. (mean ± S.E., n = 4, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5. Modified tri-gas incubator for stable hypoxic cultures.

(a) Tri-gas incubator with 4 segmented chambers. (b) Culture box with outlet filter and connector to tri-gas mixture injection tube. (c) Assembled culture boxes inside the tri-gas incubator. (d) A schematic diagram for the modified culture chamber.