April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Use of Wnt and Nodal Inhibitors to Induce Embryonic Stem Cell Differentiation Into Retinal Pigment Epithelium (RPE)
Author Affiliations & Notes
  • J. Gong
    Ophthalmology, Columbia University, New York, New York
  • H. Cai
    Ophthalmology, Columbia University, New York, New York
  • T. Y. Liu
    Ophthalmology, Columbia University, New York, New York
  • F. Mark
    Ophthalmology, Columbia University, New York, New York
  • L. Del Priore
    Ophthalmology, Columbia University, New York, New York
  • Footnotes
    Commercial Relationships  J. Gong, None; H. Cai, None; T.Y. Liu, None; F. Mark, None; L. Del Priore, None.
  • Footnotes
    Support  Research to Prevent Blindness, Robert L. Burch III Fund, Hickey’s Family Foundation and the Foundation Fighting Blindness.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1270. doi:
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      J. Gong, H. Cai, T. Y. Liu, F. Mark, L. Del Priore; Use of Wnt and Nodal Inhibitors to Induce Embryonic Stem Cell Differentiation Into Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2009;50(13):1270.

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

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Abstract

Purpose: : Embryonic stem cell (ESC) transplantation is a promising therapeutic approach for the replacement of degenerated retinal pigment epithelium (RPE) cells in patients with age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Leber`s Congenital Amaurosis (LCA). We have shown previously that culturing human ESC on human Bruch`s Membrane and Matrigel can induced RPE markers; herein we use a combination of growth factors and Wnt and Nodal inhibitors (DKK1, Lefty A and activin) to induce ESC to differentiate along RPE or retinal progenitor lines.

Methods: : Mouse ESC were grown for 9 days as aggregates in serum-free medium with DKK1, Lefty A and activin (Osakada F, Nature biotechnology 2008). Subsequently these stem cells were treated with DAPT, a Notch signal inhibitor, in a retinal differentiation medium containing retinoic acid, taurine, basic FGF and acid FGF and cultured up to 28 days on dishes coated with poly-D-lysine, laminin and fibronectin. These differentiated cells were examined under fluorescence microscopy after staining for RPE markers (Bestrophin and ZO-1) and photoreceptor markers (rhodopsin, cone opsin, Nrl and CRX).

Results: : After 24 hour treatment with Wnt and Nodal inhibitors, mouse ESC formed embryoid body aggregates in suspension. By day 7 these aggregates increased exponentially in size to form larger pigmented clumps, cells grew onto monolayers after seeding onto laminin and fibronectin coated dishes. At 28 days in retinal differentiation medium, these cells appeared to take on an RPE-like morphology and express Bestrophin and ZO-1. However, these cells expressed NRL weakly, and were immune negative for rhodopsin, cone opsin and CRX.

Conclusions: : Embryonic stem cells have the potential to differentiate into RPE when cultured onto laminin and fibronectin in the presence of growth factors and Wnt and Nodal inhibitors. Further study is required to assess the function to ESC-derived RPE under different culture conditions.

Keywords: retinal pigment epithelium • growth factors/growth factor receptors 
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