June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Retinal Pigment Epithelial Cell Differentiation in Primary Human Limbal Neurosphere Cultures
Author Affiliations & Notes
  • Samuel McLenachan
    Ocular Tissue Engineering, Lions Eye Institute, Nedlands, WA, Australia
    Centre of Ophthalmology and Visual Science, University of Western Australia, Perth, WA, Australia
  • Dana Zhang
    Ocular Tissue Engineering, Lions Eye Institute, Nedlands, WA, Australia
    Centre of Ophthalmology and Visual Science, University of Western Australia, Perth, WA, Australia
  • Fred Chen
    Ocular Tissue Engineering, Lions Eye Institute, Nedlands, WA, Australia
    Centre of Ophthalmology and Visual Science, University of Western Australia, Perth, WA, Australia
  • Footnotes
    Commercial Relationships Samuel McLenachan, None; Dana Zhang, None; Fred Chen, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1008. doi:
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    • Get Citation

      Samuel McLenachan, Dana Zhang, Fred Chen; Retinal Pigment Epithelial Cell Differentiation in Primary Human Limbal Neurosphere Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1008.

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

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Abstract
 
Purpose
 

The human corneoscleral limbus contains multipotent stem cells that can be isolated and cultured for clinical applications, such as the treatment of limbal stem cell deficiency. In culture, limbal stem cells can be induced into the neural linage to produce cells displaying the characteristics of photoreceptors. Here, we have examined the hypothesis that retinal pigment epithelial (RPE) cells can be produced from primary human limbal cultures.

 
Methods
 

Human corneoscleral rims were incubated with collagenase to facilitate the removal of the limbal epithelium (LE). LE was dissociated non-enzymatically and cultured in the presence of EGF, FGF2 and Noggin to produce floating neurospheres (LiNS). LiNS were plated on a geltrex matrix to form adherent colonies. In parallel, primary limbal cells were cultured in keratinocyte media to produce adherent LE cell monolayers. Primary LE and LiNS cultures were examined by microscopy and immunocytochemical analysis.

 
Results
 

Two different populations of neurospheres were evident in primary human LiNS cultures; pigmented and non-pigmented. Comparison of primary keratinocyte and neurosphere cultures revealed the predominance of non-pigmented LiNS in cultures with stromal keratocyte contamination. Pigmented LiNS expressed the ocular transcription factors PAX6, OTX2 and MITF, and contained cells expressing the RPE specific markers RPE65 and ZO1.

 
Conclusions
 

Culture of human limbal epithelial stem cells in the presence of EGF, FGF2 and Noggin leads to the induction of LiNS containing pigmented cells expressing the RPE cell markers MITF, RPE65 and ZOI.

 
 
Human limbal neurosphere
 
Human limbal neurosphere
 
 
Human LiNS cells immunostained for RPE65 (green) and zonus occludin-1 (red)
 
Human LiNS cells immunostained for RPE65 (green) and zonus occludin-1 (red)
 
Keywords: 701 retinal pigment epithelium • 482 cornea: epithelium • 721 stem cells  
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