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Dean Hallam, Christin Henein, Satomi Miwa, Sajjad Ahmad, Gabriele Saretzki; The effect of hypoxia on the growth of limbal stem cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):982.
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This study aims to detail the effects of hypoxia (3% oxygen) on the in vitro culture of human limbal stem cells. The hypothesis is that hypoxic conditions may be beneficial to limbal stem cell (LSC) growth, as these cells come from the neural ectoderm lineage. In addition a number of stem cells are cultured in hypoxic conditions such as embryonic, neural crest and hematopoietic stem cells. It is thought that hypoxic conditions can shield stem cells from the damaging effects of reactive oxygen species, limiting propagation of DNA damage and retaining a more quiescent state. The successful growth and expansion of limbal stem cells is important for the clinical treatment of limbal stem cell deficiency (LSCD).
Extraction of human corneal epithelial cells was performed used serial trypsinsation followed by expansion on 3T3 mouse fibroblasts. Colony forming efficiency (CFE) assays were a performed after 12 days. RT-Q-PCR was performed on primary cell cultures to detect mRNA levels. Annexin V assay was used to measure apoptosis, whilst DAPI was used to determine cell cycle state. Metabolic activity was measured using a Seahorse XFS, which detects changes in extracellular fluctuations in oxygen and protons.
CFE assays showed a decreased number of cells and colonies in hypoxia. Quantitative RT-PCR revealed that the expression of positive LSC markers and telomerase genes was higher in hypoxia. The expressions of differentiation markers were lower in hypoxia. Flow cytometry showed a greater proportion of cells cultured in hypoxia were in G0/G1phase (83.6%) compared to normoxia (79.7%). The proportion of cells which were dead or undergoing apoptosis was 22% in normoxia and 13.1% in hypoxia. Metabolic data showed that cells grown in hypoxia had a higher basal oxygen consumption and a higher oxygen capacity during the experiment whilst cells in normoxia had a lower values.
We demonstrated that human corneal epithelium stem cells cultured in hypoxic conditions may exhibit a quiescent stem cell phenotype, which may be essential for maintaining stemness by protecting cells from differentiation and apoptosis. Metabolic data also suggests that cells grown in hypoxia are more metabolically active and have a higher mitochondrial respiratory capacity than those grown in normoxia. As 3% oxygen may be closer to physiological oxygen levels and that these conditions may favour mitochondrial biogenesis.
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