April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Differentiation Of Human Embryonic Stem Cells Into Cells With Corneal Phenotypes
Author Affiliations & Notes
  • Audrey A. Chan
    Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • Martha L. Funderburgh
    Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • Katherine Davoli
    Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • James L. Funderburgh
    Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships  Audrey A. Chan, None; Martha L. Funderburgh, None; Katherine Davoli, None; James L. Funderburgh, None
  • Footnotes
    Support  EY016415, EY09368, and P30-EY08098, Research to Prevent Blindness, and Eye and Ear Foundation of PGH
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3943. doi:
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      Audrey A. Chan, Martha L. Funderburgh, Katherine Davoli, James L. Funderburgh; Differentiation Of Human Embryonic Stem Cells Into Cells With Corneal Phenotypes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3943.

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

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Abstract

Purpose: : The clarity of the cornea is a property of the highly organized extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest (NC) lineage. Derivation of keratocytes from pluripotent human embryonic stem (hES) cells would help elucidate the keratocyte developmental pathway and also open a potential for ES cell-based therapies. The purpose of this study was to identify culture conditions that induce characteristic keratocyte gene expression patterns in pluripotent human ES cells.

Methods: : hES cell line H1 was induced to neural differentiation via co-culture with PA6 fibroblast cells. Expression of NC-specific genes was followed by qRT-PCR and a sub-population of ES derived cells expressing LNGFR (CD271, p75) was isolated with magnetic sorting. LNGFR+ cells were expanded in growth media reported to induce mesenchymal or neural differentiation, and then cultured as substratum-independent pellet cultures to induce keratocyte phenotype. The resultant cells were analyzed for keratocyte gene expression using quantitative RT-PCR analysis.

Results: : hES cells co-cultured with PA6 fibroblasts undergo a rapid transient expression of genes associated with NC cells including SNAIL, NTRK3, SOX9, MSX1. Up to 33% of these cells expressed LNGFR and HNK-1 by flow cytometry at 6 days. Cells selected for LNGFR surface expression continued expression of NC genes and were free of feeder cells. LNGFR+ cells expanded in alpha MEM+FBS or in serum-free media with N2 supplement both expressed genes found in stromal progenitor cells (ABCG2, BMI1, KIT, PAX6, Nestin, NOTCH1, SIX2). In pellet culture, conditions favoring keratocyte differentiation, the cells expressed elevated keratocyte-specific markers including KERA, AQP1, B3GnT7, PTDGS, and ALDH. Expression of keratocan, a keratocyte-specific proteoglycan, was increased >10,000 fold. Markers of adult corneal stem cells PAX6, nestin, notch1, BMI1 and cKit, were decreased in the pellet-cultured cells.

Conclusions: : hES cells can be induced to differentiate into cells that express keratocyte-specific markers in vitro. Pluripotent hES cells therefore may provide a potential renewable source of tissue for the surgical treatment of severe corneal opacities.

Keywords: cornea: stroma and keratocytes • cornea: basic science • differentiation 
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