September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Validation of pluripotent stem cell derived RPE (scRPE) as a pure and renewable source for disease modeling and assay development
Author Affiliations & Notes
  • Ulrich F O Luhmann
    Pharma Research and Early Development, Ophthalmology Discovery and Biomarkers , Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Sonja Schlicht
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Faye M Drawnel
    Pharma Research and Early Development, Ophthalmology Discovery and Biomarkers , Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Silke Zimmermann
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Carolin Willburger
    Pharma Research and Early Development, Ophthalmology Discovery and Biomarkers , Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Claire Hippert
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Verena Küppers
    Pharma Research and Early Development, Ophthalmology Discovery and Biomarkers , Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Jean-Baptiste Vallier
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Jean-Philippe Carralot
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Marco Prunotto
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Mark Burcin
    Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland
  • Footnotes
    Commercial Relationships   Ulrich Luhmann, F. Hoffmann-La Roche Ltd (E); Sonja Schlicht, F. Hoffmann-La Roche Ltd (E); Faye Drawnel, F. Hoffmann-La Roche Ltd (E); Silke Zimmermann, F. Hoffmann-La Roche Ltd (E); Carolin Willburger, F. Hoffmann-La Roche Ltd (E); Claire Hippert, F. Hoffmann-La Roche Ltd (E); Verena Küppers, F. Hoffmann-La Roche Ltd (E); Jean-Baptiste Vallier, F. Hoffmann-La Roche Ltd (E); Jean-Philippe Carralot, F. Hoffmann-La Roche Ltd (E); Marco Prunotto, F. Hoffmann-La Roche Ltd (E); Mark Burcin, F. Hoffmann-La Roche Ltd (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 257. doi:
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      Ulrich F O Luhmann, Sonja Schlicht, Faye M Drawnel, Silke Zimmermann, Carolin Willburger, Claire Hippert, Verena Küppers, Jean-Baptiste Vallier, Jean-Philippe Carralot, Marco Prunotto, Mark Burcin; Validation of pluripotent stem cell derived RPE (scRPE) as a pure and renewable source for disease modeling and assay development. Invest. Ophthalmol. Vis. Sci. 2016;57(12):257.

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

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Abstract

Purpose : Impaired function and loss of the retinal pigment epithelium (RPE) impact on photoreceptor health and contribute to vision loss in degenerative retinal diseases such as age-related macular degeneration, one of the most common causes for blindness in the developed world. To identify a reliable and renewable source of human RPE cells for disease modelling and assay development we implemented a differentiation protocol for pluripotent stem cell derived RPE cells (scRPE) and characterized their biological activity and function relative to ARPE19 cells and commercially available primary human RPE under different culture and assay conditions.

Methods : To obtain RPE from pluripotent stem cells we developed a stepwise differentiation protocol based on small molecules inhibiting the Wnt and Nodal pathways during early cell development. To be assay compatible, we developed cultivation conditions to routinely expand, bank and mature scRPE. To validate their functional properties and compare scRPE to primary human RPE and ARPE-19 cells, we utilized gene expression profiling by PCR and immunocytochemistry to identify gene products that represent important functional properties of RPE. Further, we established assay conditions to test scRPE for their oxygen consumption capacity, ROS and cytokine production, cell polarization and barrier function.

Results : Under identical cultivation conditions scRPE show a similar morphology to primary RPE. PCR analysis and immunostaining show that scRPE and primary RPE express RPE specific marker genes such as ZO-1 (tight junction), RPE65 (visual cycle), CRALBP (visual cycle), Tyrosinase (melanin biosynthesis), CD68 (phagocytosis) and VEGFA (vascular endothelial cell proliferation and survival). In comparison, ARPE-19 cells clearly show differences in morphology and gene expression. By measuring oxygen consumption, cell polarization, barrier function, and stressor-induced ROS and cytokine production scRPE cells produce comparable results to primary human RPE.

Conclusions : ScRPE obtained by our differentiation protocol provide a reliable and renewable source of RPE cells that are functionally comparable to primary human RPE in several disease-relevant assay formats. This uniform source of scRPE cells will be useful for disease modelling in vitro and the development of novel assays for drug development.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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