June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Improving Human RPE Differentiation from iPS Cells: Lessons from Mouse Eye Development
Author Affiliations & Notes
  • Kapil Bharti
    NEI, National Institutes of Health, Bethesda, MD
  • Janine Davis
    NEI, National Institutes of Health, Bethesda, MD
  • Barbara Corneo
    National Eye Institute, National Institutes of Health, Bethesda, MD
  • Qin Wan
    NEI, National Institutes of Health, Bethesda, MD
  • Kiyoharu Miyagishima
    NEI, National Institutes of Health, Bethesda, MD
  • Sally Temple
    National Eye Institute, National Institutes of Health, Bethesda, MD
  • Sheldon Miller
    NEI, National Institutes of Health, Bethesda, MD
  • Footnotes
    Commercial Relationships Kapil Bharti, None; Janine Davis, None; Barbara Corneo, None; Qin Wan, None; Kiyoharu Miyagishima, None; Sally Temple, Athghin Biotech (I); Sheldon Miller, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4051. doi:
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      Kapil Bharti, Janine Davis, Barbara Corneo, Qin Wan, Kiyoharu Miyagishima, Sally Temple, Sheldon Miller; Improving Human RPE Differentiation from iPS Cells: Lessons from Mouse Eye Development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4051.

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

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Abstract

Purpose: Induced pluripotent stem (iPS) cell technology has provided the possibility of studying disease mechanisms in vitro and of developing cell-based therapy. Retinal pigment epithelium (RPE) cells can be differentiated from iPS cells, but the differentiation efficiency is variable and remains low across different patient specific iPS lines. The aim of this study is to improve iPS cell to RPE differentiation efficiency.

Methods: A previously characterized iPSC reporter line that expresses RPE-specific GFP and mouse models that express different levels of transcription factors Pax6 and Mitf were used to optimize the differentiation protocol. Several different growth factor combinations were tested for their ability to improve GFP signal, expression of PAX6, MITF, other RPE signature genes in human iPS cells differentiating towards RPE. Flow cytometry was used to analyze the GFP signal and qRT-PCR was used to analyze the expression of specific genes. Physiological assays including measurement of transepithelial resistance, transepithelial potential, and fluid flow and calcium regulation across the RPE monolayer used to authenticate fully differentiated RPE cells.

Results: The analyses of mouse models showed that PAX6 and MITF co-expression inhibits retinal fate and promotes differentiation of eye progenitors into RPE. Because the GFP expression in the reporter iPS cells is dependent on PAX6 and MITF activity, we checked the ability of FGF, BMP, WNT, SONIC, and TGF pathways to change GFP expression during iPSC to RPE differentiation. The newly optimized protocol differentiates up to 92% iPS cells into RPE-like cells, as judged by the GFP expression. This method increases, by several-fold, the expression of MITF and PAX6 in cells that are progenitors to the RPE lineage. The fully differentiated cells were authenticated using molecular and physiological assays. RPE cells generated from iPS cells using the optimized method closely resemble primary human RPE cells in terms of their molecular and physiological properties.

Conclusions: We have developed a protocol that generates fully differentiated RPE cells at a high efficiency. This protocol is easily amenable to current Good Manufacturing Practices and can be used to generate large quantities of RPE cells for high throughput screens.

Keywords: 701 retinal pigment epithelium • 721 stem cells • 687 regeneration  
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