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Roy Joseph, Om P Srivastava, Roswell R Pfister; Generation of Induced Pluripotent Stem Cells from Normal and Keratoconus Corneal Fibroblasts using Viral- and Non-Viral Methods.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):523.
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To generate induced pluripotent stem cells (iPSC) from fibroblasts of both keratoconus and normal human cornea by viral- and non-viral methods.
Primary corneal fibroblasts were generated from both normal and keratoconus corneal stroma. Both normal and keratoconus corneal fibroblasts from three individuals were reprogramed directly to pluripotent stem cells (PSC) by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, using a single polycistronic lentiviral vector co-expressing four transcription factors (Oct 4, Sox2, Klf4 and c-Myc) to yield induced pluripotent stem cells (iPSC). The non-viral method was based on the single-vector reprogramming system and was combined with a piggyBac transposon. Phase-contrast images were obtained using Olympus 1X 71 inverted microscope. These iPS cells were characterized by immunofluorescence detection (Zeiss Axioplan 2 Microscope) of stem cell markers (SSEA4, Oct4 and Sox2) and also by quantitative RT-PCR. The corneal stem cells isolated from the limbal region of both epithelium and stroma of normal corneas were used as positive controls.
We obtained 4-12 human embryonic stem cell-like colonies per 105 cells. Our phase-contrast microscopic images of iPSC from both keratoconus and normal corneas showed a good embryonic stem cell-like clone. Immunofluorescence analysis of these colonies showed that they were positive for all the three stem cell markers (SSEA4, Oct4 and Sox2) compared to the above described positive controls. Quantitative RT-PCR analysis showed 3-fold increase in the expression of stem cell markers.
We successfully reprogramed both normal and keratoconus corneal fibroblasts using single polycistronic virus co-expressing four transcription factors. These iPSC from keratoconus corneal fibroblast will be used to study the molecular mechanism of keratoconus disease. Since we have generated iPSC by using both viral- and non-viral methods, this would help us to study differences in both forms of iPSC.
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