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Katie Leigh Bales, Roy Joseph, Om P Srivastava; Reprogramming Lens Mesenchymal Cells to Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):739.
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© ARVO (1962-2015); The Authors (2016-present)
To determine a specific timeline for lens epithelial to mesenchemymal cell transition (EMT) and reprogramming of the transitioned mesenchymal cells to induced pluripotent stem cells (iPSCs) using viral vectors.
Lens epithelial cells were isolated from lenses of one-month old C57BL/6 mice and were cultured in 6-well plates in M199 medium with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were maintained for five days for mesenchymal transition. Mesenchymal cells were directly reprogramed, delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, using a single polycistronic lentiviral vector co-expressing four transcriptional factors (Oct4, Sox2, Klf4 and C-Myc) to yield iPSCs. After twenty days, clones were removed for immunohistochemical cell analysis and RNA isolation. Cells were fixed in 4% paraformaldehyde at room temperature and examined for epithelial markers (Connexin-43, E-cadherin), mesenchymal markers (Alpha smooth muscle actin), lens-specific markers (CryAB) and stem cell markers (Sox1, Oct4, SSEA4).
Based on results from immunohistochemical cell analysis and Quantitative PCR, at day zero, there was an increased expression of epithelial markers and by day two, there was an up-regulation of mesenchymal markers. Stem cell markers were not present at both the time-points. Mesenchymal cells were then reprogramed directly to iPSCs after twenty days, obtaining four to six embryonic stem cell-like colonies per 105 cells. Immunohistochemical analysis showed clones were positive for all three stem cell markers.
Transitioning of lens epithelial cells to mesenchymal cells resulted in the loss of epithelial markers and up-regulation of mesenchymal cell markers, suggesting EMT. Our data show that using a single polycistronic lentiviral vector co-expressing four transcriptional factors can successfully reprogram mesenchymal cells generated from lens epithelial cells into iPSCs.
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