April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Characteristics of Corneal Stromal Side Population Cells Isolated With DyeCycle Violet
Author Affiliations & Notes
  • Y. Du
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • N. Zurowski
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • J. L. Funderburgh
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships  Y. Du, None; N. Zurowski, None; J.L. Funderburgh, None.
  • Footnotes
    Support  NIH grant EY016415 (JLF), Research to Prevent Blindness, The Eye and Ear Foundation of Pittsburgh. JLF is a Jules and Doris Stein Research to Prevent Blindness Professor.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1776. doi:
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      Y. Du, N. Zurowski, J. L. Funderburgh; Characteristics of Corneal Stromal Side Population Cells Isolated With DyeCycle Violet. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1776.

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

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Purpose: : The ability of adult stem cells to separate as a side population (SP) in flow cytometry has been widely investigated. SP cells are obtained using an ultraviolet laser after loading cells with Hoechst 33342 dye. This procedure can be damaging to cells, and SP isolates often fail to grow in vitro. DyeCycle Violet (DCV) reagent is a cell-permeable DNA-binding dye excited by violet light recently found to produce Hoechst-like SP (Ho-SP) separations. Previously we described stem cells in human corneal stroma isolated as SP with Hoechst 33342. In the current study we compared properties of SP cells sorted by these two different methods.

Methods: : Human and bovine corneal stroma were digested by collagenase. Human stromal cells were cultured in a defined stem cell culture medium for 1-3 passages before sorting. Bovine stromal cells were stained and sorted directly after digestion. SP cells, cultured 3-4 passages after sorting, were examined for expression of ABCG2, Pax6, Notch1, Bmi1 and keratocan by qRT-PCR. Sorted SP cells were induced to differentiate to keratocyte phenotype using a pellet culture as previous reported. qRT-PCR and immunostaining were used to examine the keratocan expression.

Results: : SP profiles were similar comparing Ho-SP and DCV-SP for both human and bovine cells. However, a greater portion of human isolates were viable in vitro after DCV-SP sorting. DCV-SP formation was inhibited by fumitremorgin C indicating involvement of ABCG2 or similar transporters, but Ho-SP was more sensitive to verapamil. Both Ho-SP and DCV-SP could be passaged in vitro with similar cellular morphologies, and both expressed stem cell-related genes ABCG2, Pax6, Notch1 and Bmi1, but no keratocan. Keratocan expression was upregulated in human DCV-SP cultured as pellets.

Conclusions: : The SP from corneal stromal cells sorted by violet-excited flow cytometer using DCV reagent is similar to the SP sorted by UV-laser using Hoechst 33342 but has higher viability. The data suggests that DCV efflux identifies the same stem cell population as Hoechst efflux in corneal stromal cells. Thus DCV can be a substitute for Hoechst dye in isolation of corneal stromal stem cells.

Keywords: cornea: stroma and keratocytes • flow cytometry • cell survival 

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