Abstract
Purpose:
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.
Methods:
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).
Results:
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.
Conclusions:
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.
Keywords: 512 EMT (epithelial mesenchymal transition) •
721 stem cells •
533 gene/expression