June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
A Tri-phasic Developmentally Guided Differentiation Protocol for Efficient Generation of Functional Retinal Pigment Epithelium from Induced Pluripotent Stem Cells
Author Affiliations & Notes
  • Fnu Ruchi
    NEI, NIH, Bethesda, MD
  • Janine Davis
    NEI, NIH, Bethesda, MD
  • Balendu Shekhar Shekhar Jha
    NEI, NIH, Bethesda, MD
  • Vladimir Khristov
    NEI, NIH, Bethesda, MD
  • Juliet Hartford
    NEI, NIH, Bethesda, MD
  • Fang Hua
    NEI, NIH, Bethesda, MD
  • Qin Wan
    NEI, NIH, Bethesda, MD
  • Kapil Bharti
    NEI, NIH, Bethesda, MD
  • Footnotes
    Commercial Relationships Fnu Ruchi, None; Janine Davis, None; Balendu Shekhar Jha, None; Vladimir Khristov, None; Juliet Hartford, None; Fang Hua, None; Qin Wan, None; Kapil Bharti, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1841. doi:
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      Fnu Ruchi, Janine Davis, Balendu Shekhar Shekhar Jha, Vladimir Khristov, Juliet Hartford, Fang Hua, Qin Wan, Kapil Bharti; A Tri-phasic Developmentally Guided Differentiation Protocol for Efficient Generation of Functional Retinal Pigment Epithelium from Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1841.

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

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Purpose: Induced pluripotent stem (iPS) cell technology is an important tool for studying disease mechanism and for developing cell-based therapies. One of the potential applications of iPS cell technology is the generation of retinal pigment epithelium (RPE), a monolayer of cells located posterior to the retinal photoreceptors. Degeneration of these cells leads to blinding eye diseases like age-related macular degeneration that is a major cause of blindness in US. In this study, we aim to develop an efficient and reproducible protocol to differentiate iPS cells to functional RPE monolayer.

Methods: A reporter iPS cell line expressing RPE-specific GFP and constitutive RFP was used to optimize the differentiation protocol. Differentiation of iPS cells from EB stage to RPE was carried out using developmentally guided protocol mimicking stages of in vivo RPE development. This triphasic protocol starts with the commitment of iPS cells towards RPE-primed neuroectoderm using a dual SMAD, canonical WNT, and FGF inhibition. Committed RPE cell differentiation requires an activation of Canonical WNT and TGF-signaling pathways and maturation of RPE cells requires a down regulation of canonical WNT pathway. Differentiation efficiency was determined using GFP expression. gene expression, and pigmentation. Reproducibility of the protocol was determined using several different healthy and patient-derived iPS cell lines. Functionality of RPE monolayers was determined using electrophysiology and phagocytosis assays.

Results: The first phase of the protocol significantly increases the expression of eye-field transcription factors PAX6, RAX, OTX1, and SIX3; GFP expression significantly increases by using a combination of dual-SMAD and FGF inhibition. In the second phase, use of canonical WNT and ACTIVIN A further increases GFP levels in the cells and increases the expression of RPE-specific transcription factors PAX6, MITF, and OTX2 generating RPE committed cells. In the third phase cells mature and start expressing maturation RPE makers like RPE65.

Conclusions: The use of reporter line has helped us in improving the iPS cell to RPE differentiation efficiency. Combined the three phases of differentiation result in more than 98% RPE differentiation efficiency and generate fully differentiated and functional RPE cells.


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