6533b85efe1ef96bd12c0584

RESEARCH PRODUCT

The importance of culturing primary cells under physiological conditions: proliferation, senescence, pluripotency

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Mesenchymal stem cells (MSCs), such as human dental pulp stem cells (hDPSCs), are currently a source for cell therapy. However, cell therapy protocols require 10–400 million cells per treatment, and consequently, they need to be expanded in vitro before implantation, with the inconvenience that MSCs undergo senescence following a certain number of cell expansion passages, loosing their stem cell qualities. Ambient oxygen tension (21% pO2) is normally used for in vitro culture, but physiological levels in vivo range between 3% and 6% pO2. We previously demonstrated that hDPSC proliferation rate is significantly lowered at 21% pO2 due to enhanced oxidative stress, which led to the activation of p38/p21/NRF-2 pathway, upregulating antioxidant defenses. Moreover, long-term in vitro culture of hDPSCs at 21% pO2 caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and lysil oxidase expression, which is mediated by p16INK4a pathway. This was accompanied by a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16INK4a and BMI-1 might play a role in the oxidative stress- associated premature senescence. For all these reasons, we show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence.