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Hui Lin, Benjamin Botsford, Samuel C Yiu; Genomic DNA Global Methylation of Primary Cultured Rabbit Lacrimal Gland Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5507.
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© ARVO (1962-2015); The Authors (2016-present)
DNA methylation is essential for normal development and is associated with a number of key cellular processes including gene expression, genomic imprinting, X-chromosome inactivation, suppression of repetitive elements, chromatin modification and carcinogenesis. Global DNA methylation may potentially be as a marker to differentiate the progenitor cells from the more differentiated ones. Here we investigate the global methylation status of primary cultured rabbit lacrimal gland (LG) progenitor cells.
The LG epithelial cells were isolated from ligation and non-ligated control groups, as reported previously. Thereafter, the progenitor cells were expanded in serum free medium with single cell clonal assay. NanoMeth (Harborgen Biotechnology, MD, USA), a novel kit with quantitative polymerase chain reaction (qPCR) based technology, was used to measure global genomic DNA methylation at different passages. The epithelial progenitor cell marker ∆Np63, gene expression was accessed by real-time PCR was performed to detect the cell proliferation capability in certain passage of cells of ligation group. Rabbit dermal fibroblast and human pancreatic epithelial cell line (PANC-1) was cultured and accessed for the global methylation ratio as controls.
LG epithelial progenitor cells isolated from ligated samples were successfully passaged through P10 and the progenitor cells from non-ligated control group can be passaged to P6. The global methylation percentage of P3 cells in ligation group was 18.51%, whereas 24.66% in control group and 29.05% in non-selected primary LG epithelial cells. Moreover, for LG progenitor cells from ligated samples, the global methylation ratio in P2 and P10 was 21.04% and 18.03%. The ∆Np63 gene expression level of LG progenitor cells in the ligated group increased from P1 to P10, and was higher compared to control group. Rabbit fibroblast underwent senescence gradually after 3 passages, and the global methylation ratio increased with every passage. The PANC-1 cells had stable global methylation rate within passages.
The rabbit LG epithelial progenitor cells, expanded in vitro, exhibit different global methylation patterns in early and late passages, compared to immortalized cell line or gradually senescent fibroblast. Global methylation ratio may serve as an indicator of cell aging and proliferation capability of primary cells.
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