Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

Abstract

The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O₂). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21^WAF1/CIP1 expression in the G₀/G₁ phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75^NTR and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G₀/G₁ phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21^WAF1/CIP1 and p16^INK4A and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

Noninvasive measurement of cell/colony motion using image analysis methods to evaluate the proliferative capacity of oral keratinocytes as a tool for quality control in regenerative medicine

“…Primary oral mucosa keratinocyte cultures were established, and cells were serially subcultured as previously described. 23 Briefly, after excess blood on the tissue specimen was removed using a scalpel, the specimen was transferred to a 0.025% trypsin/EDTA solution (Thermo Fisher Scientific, Waltham, MA, USA) containing 1.5% Antibiotic-Antimycotic (Thermo Fisher Scientific) and soaked for approximately 16 h at room temperature. Oral mucosa keratinocytes were mechanically dissociated from the underlying connective tissue in a 0.0125% defined trypsin inhibitor (Thermo Fisher Scientific), resuspended in ‘complete’ EpiLife ® supplemented with EpiLife Defined Growth Supplements (Thermo Fisher Scientific), 0.06 mM Ca 2+ , plated at a density of 3.0–4.0 × 10 5 cells in a 35 mm dish (Eppendorf) with complete EpiLife ® medium, and fed every other day.…”

Metabolomic Alteration of Oral Keratinocytes and Fibroblasts in Hypoxia

“…Glycolysis and PPP dominantly produce energy and suppress the generation of ROS, which is an important mediator of cell damage and the cell death process, and it maintains the quiescent state as well. We previously reported that ROS generation was suppressed under hypoxia in oral keratinocytes [ 14 ]. Nonetheless, the proliferation of oral keratinocytes increased at 2% and 0.5% oxygen concentrations [ 14 ].…”

“…We previously reported that ROS generation was suppressed under hypoxia in oral keratinocytes [ 14 ]. Nonetheless, the proliferation of oral keratinocytes increased at 2% and 0.5% oxygen concentrations [ 14 ]. A decrease of G6P was seen in hypoxic oral fibroblasts.…”

“…The influence of oxygen concentrations on cancer and wound healing in oral mucosa has been examined [ 12 , 13 ]; however, no report on the oxygen concentration in vivo has been published. Since the culture of oral mucosal epithelial cells under hypoxic conditions suppresses differentiation and senescence and promotes colony formation, hypoxic exposure is beneficial for applications in regenerative medicine, similar to other cells [ 14 ].…”

Improvement of Antioxidant Defences in Keratinocytes Grown in Physioxia: Comparison of 2D and 3D Models

“…A consequence of oxygen metabolism in cells is the production of reactive oxygen species (ROS), and their amount depends on the environmental oxygen availability [5][6][7][8][9]. A basal intracellular ROS level is required for cell life and physiological functions as these species are involved in cell proliferation and differentiation, in vascular tone and apoptosis regulation, and also in the defence against microorganisms [10].…”