April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
CD271 is a Cell-Surface Marker of Keratocyte Progenitor Cells
Author Affiliations & Notes
  • J. L. Funderburgh
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • M. M. Mann
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • M. L. Funderburgh
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • D. S. Roh
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • Y. Du
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships  J.L. Funderburgh, None; M.M. Mann, None; M.L. Funderburgh, None; D.S. Roh, None; Y. Du, None.
  • Footnotes
    Support  NIH Grants EY016415 30-EY08098. Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2952. doi:
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      J. L. Funderburgh, M. M. Mann, M. L. Funderburgh, D. S. Roh, Y. Du; CD271 is a Cell-Surface Marker of Keratocyte Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2952.

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Abstract

Purpose: : A number of studies have identified populations of stem and progenitor cells in the stroma of mammalian corneas, however the cell-surface antigens of these cells are not well defined. Recently studies have used cell surface expression of CD271 (p75NGFR) to identify and select populations of mesenchymal stem cells from several adult tissues. The same antigen was also used to identify neural crest stem cells in cultures of differentiating embryonic stem cells. Since stromal keratocytes are derived from neural crest cells, we initiated this study to investigate the possibility that CD271 was present on progenitor cells in the corneal stroma.

Methods: : CD271 expression was determined by flow cytometry on fresh collagenase-isolated cells from bovine and human corneas. Cells expressing CD271 were isolated by MACS chromatography and cultured at clonal density in low-serum media to expand progenitor cells or at high density in serum-free media with insulin and ascorbate to induce keratocyte differentiation. Cell phenotype and gene expression were determined by qPCR, by immunoblotting, and by immunostaining.

Results: : Greater than 90% of bovine and 60% of human freshly-isolated stromal cells exhibited cell-surface CD271 detectable by flow cytometry. Neither endothelial nor epithelial cells showed CD271 expression. MACS chromatography separated bovine stromal cells into two populations differing by 5-fold in the level of CD271 expression. The high expressing cells (CD271Hi) showed higher levels of clonogenicity and also expressed higher levels of keratan sulfate in conditions favoring differentiation compared to low expressing cells. CD271Hi cells plated at clonal density and expanded through more than ten population doublings maintained an ability to differentiate to keratocytes and myofibroblasts.

Conclusions: : A large proportion of adult mammalian stromal cells express a marker associated with neural crest stem cells. High levels of expression of CD271 are associated with the keratocyte progenitor phenotype in these cells. These results suggest that many stromal cells maintain progenitor potential in the adult cornea.

Keywords: cornea: stroma and keratocytes • development • flow cytometry 
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