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Kiran Srivastava, Roy Joseph, Om P Srivastava, ; Reprogramming Lens Mesenchymal Cells to Induced Pluripotent Stem Cells and also Differentiating Stem Cells to Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2656.
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© ARVO (1962-2015); The Authors (2016-present)
To reduce the time of reprograming lens mesenchymal cells to induced pluripotent stem cells (iPSC) and also to differentiate iPSC to epithelial cells.
Lens epithelial cells were isolated from 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 in the media until mesenchymal transition. The mesenchymal cells were then reprogramed directly 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 Myc) to yield induced pluripotent stem cells. The lens mesenchymal cells were virally transduced repeatedly twice at 24 h intervals. The clones were removed for immunohistochemical cell analysis. 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 and Tra 60). The differentiation of iPS cells was done according to a method described previously (PLOS one,2012, e32612)
By increasing the number of genetic transduction, we were able to reduce the time for generating iPSC from lens mesenchymal cells. Mesenchymal cells reprogramed directly to iPSCs after 8 to 10 days, whereas previously when genetic transduction was done once, it took 20 days to generate iPSC from lens mesenchymal cells (ARVO 2014, Abstract no. 517). We obtained four to six embryonic stem cell-like colonies per 105 cells. Immunohistochemical analysis showed that clones were positive for all the four above described stem cell markers.
Our results show that ectopic expression of transcription factors can successfully reprogram mesenchymal cells (generated from lens epithelial cells) into induced pluripotent stem cells. We were able to reduce the time needed for generating iPSC with a minimal loss of cells.
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