April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Predicting Regenerative Potential of Corneal Stromal Stem Cells
Author Affiliations & Notes
  • James L Funderburgh
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, PA
  • Martha L Funderburgh
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, PA
  • Mary Mann
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, PA
  • Footnotes
    Commercial Relationships James Funderburgh, None; Martha Funderburgh, None; Mary Mann, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5132. doi:
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      James L Funderburgh, Martha L Funderburgh, Mary Mann; Predicting Regenerative Potential of Corneal Stromal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5132.

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

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Purpose: Mesenchymal stem cells from limbal stroma (corneal stromal stem cells, CSSC) restore corneal transparency and prevent scarring in mouse models. CSSC therefore present the potential for use in corneal regenerative therapy. Isolated CSSC lines, however, vary in stem cell potency, and currently few tools are available to predict the differentiation potential of cultured CSSC. This study attempted to identify phenotypic properties that correlate with the regenerative potential of CSSC. Such markers could provide a valuable quality control metric when translating stem cell applications to human therapy.

Methods: CSSC were isolated by collagenase from limbal biopsies of corneal rims after DSEAK or PK. Cells were cultured in SCGM, a low-serum medium in plastic dishes (PMID16051989). Primary cultures were expanded at clonal density (<1000 cells/cm sq ) and used at passage 2. Clones were isolated from 250 cells plated in 10 cm dishes in SCGM. Gene expression was determined by qPCR and Nanostring nCounter. Keratocyte differentiation was induced in serum-free medium (PMID23557404) on collagen-coated plates.

Results: Early passage CSSC expressed a variety of genes associated with mesenchymal, neural, and pluripotent stem cells. Culture on Matrigel in serum-free media produced little difference in this pattern of gene expression, but culture as spheres upregulated pluripotent markers. Serial passage of CSSC resulted in a decrease in clonogenic potential of the CSSC from 40% to 0% within 6 passages (20 population doublings). Stem cell marker expression was not lost during expansion, except for that of PAX6, a transcription factor essential for ocular development and expressed in neural progenitor cells . Formal cloning of CSSC revealed a heterogeneous potential for sub-cloning and expansion similar to that of the holoclones and meroclones characteristic of epithelial stem cells.

Conclusions: In spite of expansion at clonal density, mesenchymal stem cells from corneal stroma represent a heterogeneous population with mixed potential for differentiation. This potential is rapidly lost on expansion. Our results suggest that obtaining a stabile, homogeneous stem cell reagent with predictable properties will require multiple rounds of clonal selection. The best predictor of keratocyte differentiation potential in these cells appears to be expression of the transcription factor PAX6.

Keywords: 484 cornea: stroma and keratocytes • 721 stem cells • 500 differentiation  

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