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Roy Joseph, Om P Srivastava, Roswell R Pfister; Differentiation of Normal Human Corneal Induced Pluripotent Stem Cells to Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2367.
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© ARVO (1962-2015); The Authors (2016-present)
To differentiate human corneal induced pluripotent stem cells (iPSC) to retinal progenitor cells.
Normal human corneal fibroblasts from three donors 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 Myc) to yield induced pluripotent stem cells (iPSC). Phase-contrast images were obtained using our Olympus 1X71 inverted microscope. These iPS cells were immunofluorescentally characterized for stem cell markers (SSEA4, Oct4 and Sox2). The iPS cells were grown in embryoid medium (EM) [(DMEM F-12 medium containing 10% knockout serum replacement, 2% B27 supplement, 1% N2 supplement, 1% L-glutamine, 1% 100X NEAA, 1% penicillin/streptomycin, 1 ng/ml noggin, 1 ng/ml DKK-1, 1 ng/ml IGF-1 and 0.5 ng/ml bFGF], and plated at a density of 50 cell clumps/cm2 in ultralow attachment plates. Cell clumps were cultured for 5 days, and then embryoid bodies were removed and differentiated in EM +10 ng/ml noggin, 10 ng/ml DKK-1, 10 ng/ml IGF-1 and 1 ng/ml bFGF (Differentiation medium 1) in 6-well culture plates coated with poly-D-lysine, collagen, laminin and fibronectin. Media were changed every other day for 10 days with Differentiation media 1, then every other day for an additional 18 days with Differentiation media 1 +10 nm DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a γ- secretase inhibitor).
Immunofluorescence analysis of the colonies showed that they were positive for all the three stem cell markers (SSEA4, Oct4 and Sox2). The iPS cells were grown to differentiate for 33 days, and then immunocytochemical analysis of the cells showed expression of retinal photoreceptor markers, recoverin and rhodopsin.
We successfully reprogramed normal human corneal fibroblasts using single polycistronic virus co-expressing four transcription factors. We were also able to differentiate the iPSC to retinal precursor cells which expressed photoreceptor markers, rhodopsin and recoverin. These retinal precursor cells are a valuable source which could be used for transplantation in retinal degenerative diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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