April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Reprogramming Lens Mesenchymal Cells to Induced Pluripotent Stem Cells
Author Affiliations & Notes
  • Katie Leigh Bales
    University of Alabama Birmingham, Birmingham, AL
  • Roy Joseph
    University of Alabama Birmingham, Birmingham, AL
  • Om P Srivastava
    University of Alabama Birmingham, Birmingham, AL
  • Footnotes
    Commercial Relationships Katie Bales, None; Roy Joseph, None; Om Srivastava, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 739. doi:
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    • Get Citation

      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)

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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  
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