The overall goal was to generate an epithelial-mesenchymal transition (EMT) model using lens epithelial cells-induced pluripotent stem cells to elucidate EMT-regulatory factors during posterior capsular opacification (PCO). For this purpose, the mouse lens epithelial cells-derived mesenchymal cells were reprogrammed to induced pluripotent stem cells (iPSC) and differentiated to lens epithelial cells to be used to determine regulatory factors during EMT. Lens epithelial cells from one-month-old C57BL/6 mice were transitioned to mesenchymal cells in culture, and were reprogrammed to iPSC by delivering reprogramming factors in a single polycistronic lentiviral vector (co-expressing four transcription factors, Oct 4, Sox2, Klf4, and Myc). iPSC were differentiated to epithelial cells by a three-step process using noggin, basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and Wnt-3. At various time points, the cells/clones were immunocytochemically analyzed for epithelial cell markers (Connexin-43 and E-cadherin), mesenchymal cell markers (Alpha-smooth muscle actin), stem cell markers (Sox1, Oct4, SSEA4 and Tra60) and lens-specific epithelial cell markers (αA- and βA3/A1-crystallins). By increasing the number of genetic transductions, the time needed for generating iPSC from lens mesenchymal cells was reduced, successfully reprogrammed epithelial/mesenchymal cells into iPSC, and retransformed iPSC into lens epithelial cells by the growth factors' treatment. The epithelial cells could serve as a model system to elucidate regulatory factors involved during EMT to therapeutically stop it.
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| http://dx.doi.org/10.1016/j.bbrep.2019.100696 | DOI Listing |
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