Activation of p38 p21 and NRF-2 Mediates Decreased Proliferation of Human Dental Pulp Stem Cells Cultured under 21 O2

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHuman mesenchymal stem cells (hMSCs) have a therapeutic potential in tissue engineering and regenerative medicine (), mostly due to immunologic properties () and their ability to differentiate into cardiovascular or neuronal cells () among others.Human dental pulp stem cells are mesenchymal cells derived from the neural crest, which have been already proved to regenerate tissue in oral inflammatory diseases (). These cells can be obtained from permanent and deciduous pulp tissue, which is easily available from teeth after extraction without ethical issues. As previously mentioned, they have a potential role for clinical use either immediately after isolation, or for use in stem cell banking. Therefore, it is highly important to obtain and culture them under the best possible conditions.The in vitro culture of MSCs has been routinely carried out under the ambient oxygen tension (18%–21% O2) (). However, in vivo these cells are not exposed to such a hyperoxic environment (). Depending on the cell type, the local oxygen tension in MSCs niches varies between 1% and 7% O2 in bone marrow () and between 10% and 15% O2 in the adipose tissue (). Although values of 3% to 6% O2 (20–40 mmHg) in adult organs and tissues have been reported (), the actual oxygen concentration in situ depends predominantly on the vascularization of the tissue and its metabolic activity (). The dental pulp has a relatively high blood flow. It is estimated to be 40–50 ml/min/100 g of pulp tissue in a mature tooth (). This flow is relatively high, compared to that of other oral tissues and skeletal muscle ().Previous studies have shown the negative impact of the ambient oxygen tension (21% O2) on the physiology of stem cells, e.g., neuronal (), bone marrow (), umbilical cord (), or adipose tissue (href="#bib9" rid="bib9 bib26" class=" bibr popnode">Efimenko et al., 2011; Kim et al., 2012). Although reduced rates of cell proliferation have been observed during culture under 21% O2, an oxygen tension that causes oxidative stress, the underlying molecular mechanisms have not been investigated in a systematic manner.The aim of this study was to determine the growth rate of human dental pulp stem cells (hDPSCs) under physiological oxygen tension (3%), and to investigate the cell signaling pathways underlying decreased stem cell proliferation during routine culture under ambient oxygen tension (21%). We show that oxidative stress is responsible for the low proliferation rate under ambient oxygen tension and describe the signaling pathway linking oxidative stress with proliferation of hDPSCs. We demonstrate that oxidative stress leads to the sequential activation of p38 MAPK, p21, and the Nuclear factor erythroid 2-related factor 2 (NRF-2) antioxidant defense pathway (href="#bib21" rid="bib21" class=" bibr popnode">Ishii et al., 2004). A practical consequence is that incubation with Trolox, a water-soluble analog of vitamin E (href="#bib41" rid="bib41" class=" bibr popnode">Raspor et al., 2005), or with an inhibitor of p38 (SB203580) restores high proliferation rate of hDPSCs even if cultured under 21% O2.