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Derivation, characterization and retinal differentiation of induced pluripotent stem cells

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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.

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Acknowledgements

This study was supported by grants from the Department of Biotechnology, India, Champalimaud Foundation, Portugal, and the Hyderabad Eye Research Foundation, INDIA. We acknowledge the animal house facility at the Centre for Cellular and Molecular Biology, Hyderabad, India, for providing the animals used in the study. We are grateful to Prof Jyostna Dhawan for her guidance and generous gifts of some of the plasmid constructs used in the study. We also thank Prof D Balasubramanian and Dr Chitra Kannabiran for their inputs and critical evaluation of the manuscript. A part of this work was presented as a poster at the ARVO annual meeting held at Fort Lauderdale, Florida, USA, in May 2011.

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Authors and Affiliations

  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

    Subba Rao Mekala, Vasundhara Vauhini, Savitri Maddileti, Subhash Gaddipati & Indumathi Mariappan

  2. Centre for Cellular and Molecular Biology, Hyderabad, India

    Usha Nagarajan

  3. Stowers Institute for Medical Research, Kansas City, Missouri, USA

    Usha Nagarajan

Authors
  1. Subba Rao Mekala
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  2. Vasundhara Vauhini
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  3. Usha Nagarajan
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  4. Savitri Maddileti
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  5. Subhash Gaddipati
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  6. Indumathi Mariappan
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Corresponding author

Correspondence to Indumathi Mariappan.

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Corresponding editor: Satish Kumar

[Mekala SR, Vauhini V, Nagarajan U, Maddileti S, Gaddipati S and Mariappan I 2013 Derivation, characterization and retinal differentiation of induced pluripotent stem cells. J. Biosci. 38 1–12] DOI 10.1007/s12038-012-9296-1

Subba Rao Mekala and Vasundhara Vauhini contributed equally to this work.

†Contributed equally to this work.

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Mekala, S.R., Vauhini, V., Nagarajan, U. et al. Derivation, characterization and retinal differentiation of induced pluripotent stem cells. J Biosci 38, 123–134 (2013). https://doi.org/10.1007/s12038-012-9296-1

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  • DOI: https://doi.org/10.1007/s12038-012-9296-1

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