April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Generation of Retinal Pigment Epithelial Cells from Human Induced Pluripotent Stem Cells
Author Affiliations & Notes
  • M. Lehmann
    Cell Biology,
    The Scripps Research Institute, La Jolla, California
  • T. U. Krohne
    Cell Biology,
    The Scripps Research Institute, La Jolla, California
  • D. F. Friedlander
    Cell Biology,
    The Scripps Research Institute, La Jolla, California
  • A. L. Dorsey
    Cell Biology,
    The Scripps Research Institute, La Jolla, California
  • W. Li
    Chemistry,
    The Scripps Research Institute, La Jolla, California
  • S. Ding
    Chemistry,
    The Scripps Research Institute, La Jolla, California
  • M. Friedlander
    Cell Biology,
    The Scripps Research Institute, La Jolla, California
  • Footnotes
    Commercial Relationships  M. Lehmann, None; T.U. Krohne, None; D.F. Friedlander, None; A.L. Dorsey, None; W. Li, None; S. Ding, None; M. Friedlander, None.
  • Footnotes
    Support  Grants from the National Eye Institute ( EY11254) and the MacTel Foundation to MF; TUK is supported by a fellowship from the German Research Foundation (KR 2863/6-1).
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2642. doi:
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      M. Lehmann, T. U. Krohne, D. F. Friedlander, A. L. Dorsey, W. Li, S. Ding, M. Friedlander; Generation of Retinal Pigment Epithelial Cells from Human Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2642.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The recent development of technologies capable of producing induced pluripotent stem cells (IPS) from adult somatic tissues may permit their use to generate autologous retinal pigment epithelium (RPE) grafts for the treatment of diseases like age-related macular degeneration (AMD). Current reprogramming techniques require retroviral transduction of four factors that include oncogenes such as c-Myc. Given the associated risk of tumor formation, alternative technologies that eliminate the use of these factors would be advantageous. We evaluated two and four factor-derived human IPS for their capacity to differentiate into RPE cells.

Methods: : IPS were generated from human fibroblasts (IMR-90) using standard four factor lentiviral transduction (Oct4, Klf4, Sox2, c-Myc) or alternatively, from primary human epidermal keratinocytes by transduction with only two factors (Oct4, Klf4) and additional treatment with small molecules (CHIR99021, tranylcypromine). Human embryonic stem cells (H1) were used for comparison. Cells were subjected to a variety of different culture conditions, and RPE cell differentiation was assessed by morphological, molecular, and functional parameters.

Results: : RPE cells could be differentiated from four factor-derived IPS and embryonic stem cells. Cells exhibited polygonal morphology and strong pigmentation, formed epithelial monolayers with intracellular tight junctions (ZO-1 positive), and expressed RPE-specific markers (RPE65, CRALBP, bestrophin). Moreover, cells phagocytosed photoreceptor outer segments and demonstrated apical-to- basolateral fluid transport in vitro. Cells were expanded over several passages and quickly regained RPE cell morphology after reaching confluency. Two factor-derived human IPS were also generated from somatic cells, differentiated into pigmented cells, and analyzed for RPE characteristics.

Conclusions: : We demonstrate the differentiation of somatic human cell-derived IPS into cells with RPE-specific morphology and function. Further optimization of differentiation efficiency is crucial for future therapeutic application of IPS-derived RPE cells as autologous grafts in diseases like AMD.

Keywords: retinal pigment epithelium • age-related macular degeneration • differentiation 
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