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    • Keywords


      Induced pluripotent stem cells (iPSCs); mouse embryonic fibroblasts (MEFs); retinal pigmented epithelium (RPE)

    • Abstract


      Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

    • Author Affiliations


      Subba Rao Mekala1 Vasundhara Vauhini1 Usha Nagarajan2 3 Savitri Maddileti1 Subhash Gaddipati1 Indumathi Mariappan1

      1. Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, L.V. Prasad Eye Institute, Hyderabad, India
      2. Centre for Cellular and Molecular Biology, Hyderabad, India
      3. Stowers Institute for Medical Research, Kansas City, Missouri, USA
    • Dates

  • Journal of Biosciences | News

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      Posted on July 25, 2019

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