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Indumathi Mariappan, Subbarao Mekala, Vasundhara Vauhini, Savitri Maddileti, Subhash Gaddipatti; Characterization and Differentiation of Induced Pluripotent Stem Cells (iPSCs) Towards Retinal Lineages. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2219.
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© ARVO (1962-2015); The Authors (2016-present)
To derive and characterize induced pluripotent stem cell (iPSC) lines and to establish a protocol for differentiating them into retinal cell types.
Recombinant retroviruses carrying the genes for the mouse Oct4, Sox2, Klf4 and cMyc were used for infecting the mouse embryonic fibroblasts (MEFs) and the transduced cells were cultured with the mouse ES medium supplemented with LIF on mitotically inactivated MEF feeders. The reprogrammed colonies showing ES-like morphology were picked up, expanded and characterized by immuno cytochemistry, fluorescence activated cell sorting and by RT-PCR for marker gene expression, authenticated by RAPD fingerprinting and assessed the methylation status of promoter regions of pluripotency genes to confirm successful reprogramming. Differentiation was initiated by forming embryoid bodies (EBs) in suspension upon withdrawal of growth factors and was subsequently maintained as adherent cultures in differentiation medium supplemented with RPE conditioned medium.
Using retroviral methods, we could derive mouse iPSCs at an efficiency of 0.01%. Out of the total 12 clones derived, 4 clones were expanded beyond 20 passages and one of the clones (4F3) was completely characterized. The endogenous copies of all the four transgenes were upregulated in this clone and also expressed the pluripotent stem cell markers like the Oct4, Nanog, SSEA1 and the alkaline phosphatase enzyme. RAPD fingerprinting confirmed its genotype to be identical to the parental MEFs. The hypomethylated status of the nanog gene promoter confirmed the successfully reprogrammed status. The differentiated miPS cells in the embryoid bodies showed commitment towards all three lineages and the expression of different germ layer specific marker genes were confirmed by RT-PCR. On extracellular matrix coated dishes, the cells from the differentiating EBs migrate out and grow as adherent cells and patches of weakly pigmented cells with typical cobble-stone phenotype similar to the RPE cells were observed after being in culture for about 6-8 weeks time. These cells were found to be positive for ZO-1 and also had phagocytic activity as observed by the uptake of 1 µM size FITC labeled latex beads. Further characterization of the differentiated cells using cell type specific markers is in progress.
The mouse iPSC lines generated by us behaved like ES cells in terms of their stemness and pluripotency and were capable of differentiating into RPE-like cells in vitro.
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