Role of p16INK4a and BMI-1 in oxidative stress-induced premature senescence in human dental pulp stem cells

Abstract

Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3-6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and increased lysil oxidase expression, which is mediated by p16^INK4a pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16^INK4a and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO₂ to retain their stemness characteristics and to delay senescence.

Keywords: Aging; Oxygen tension; Regenerative medicine.

Graphical abstract

Fig. 1

Oxidative stress related parameters in hDPSCs cultured at 21% or 3% oxygen tension along passages. (A) ROS levels measured by dihydrorhodamine-123 (DHR123), (B) Mitochondrial membrane potential levels measured by tetramethylrhodamine methyl ester (TMRM), (C) Lipid oxidation levels measured by malondialdehyde (MDA) and (D) protein oxidation levels. The data are shown as means±SD (n=5). The statistical significance is expressed as *p<0.05; ***p<0.001 versus 3% pO₂.

Fig. 2

Antioxidant genes expression in hDPSCs cultured at 21% or 3% oxygen tension. Manganese superoxide dismutase (MnSOD) levels, glutathione peroxidase (GPx) levels and catalase levels. The data are shown as means±SD (n=5). The statistical significance is expressed as ***p<0.001 versus 3% pO₂.

Fig. 3

Oxidative stress induces premature senescence in hDPSCs during long term culture at 21% oxygen tension. (A) Number of passages reached (upper pannel) and survival curve (lower pannel), (B) β-galactosidase activity measured by fluorescein di-β-_D-galactopyranoside (FDG) load, (C) LOXL1 and (D) LOXL2 relative mRNA expression levels. The data are shown as means±SD (n=5). The statistical significance is expressed as ***p<0.001 versus 3% pO₂.

Fig. 4

Oxidative stress-induced premature senescence. Correlation with p16^INK4aand p14^ARF. (A) p14^ARF mRNA levels, (B) p16^INK4a mRNA levels and (C) p16^INK4a mRNA levels when treatment with 50 μM Trolox. The data are shown as means ±SD (n=5). The statistical significance is expressed as ***p<0.001 versus 3% pO₂ and ^###p<0.001 versus 21% pO₂+Trolox.

Fig. 5

Pluripotency markers in hDPSCs cultured at 21% vs 3% oxygen tension along passages. (A) OCT4, SOX2, KLF4 and C-MYC mRNA levels and (B) TET1 mRNA levels relative expressions. The data are shown as means±SD (n=5). The statistical significance is expressed as *p<0.05; **p<0.01; ***p<0.001 versus 3% pO₂.

Fig. 6

BMI-1expression level in hDPSCs cultured at 21% vs 3% oxygen tension along passages. (A) BMI-1 protein levels, and (B) representative western-blot images of BMI-1 protein levels. The data are shown as means ±SD (n=5). The statistical significance is expressed as *p<0.05; ***p<0.001 versus 3% pO₂.

Fig. 7

BMI-1knockdown effect onSOX2andOCT4expression in hDPSCs cultured at 21% vs 3% oxygen tension. (A) SOX2 mRNA expression levels and (B) OCT4 mRNA expression levels. The data are shown as means±SD (n=3). *p<0.05; **p<0,01; ***p<0.001.

Fig S1

Representative images of the morphological changes in human dental pulp stem cells during long term culture at 21% (lower panels) or 3% oxygen tension (upper panels).

Fig S2

BMI-1expression in dental pulp stem cells cultured at 21% vs 3% oxygen tensión at passage 5. (A) Gene and (B) protein expression levels in hDPSCs cultured at 3%, 21% and siBMI-1 21% pO2. The data are shown as means ±SD (n=3) The statistical is expressed as ***p<0.001.