June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Assessing the Potential of Stem Cells to Regenerate Stromal Tissue
Author Affiliations & Notes
  • James L Funderburgh
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Martha L Funderburgh
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Mary Mann
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Irona Khandaker
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Golnar Shojaati
    Ophthalmology, Univ of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
    Ophthalmology, University Hospital Zurich, Zurich, Switzerland
  • Footnotes
    Commercial Relationships   James Funderburgh, None; Martha Funderburgh, None; Mary Mann, None; Irona Khandaker, None; Golnar Shojaati, None
  • Footnotes
    Support  DoD Award: MR130197, NIH Grant EY016415, P30-EY008098, Research to Prevent Blindness, Eye and Ear Foundation of Pittsburgh
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1425. doi:
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    • Get Citation

      James L Funderburgh, Martha L Funderburgh, Mary Mann, Irona Khandaker, Golnar Shojaati; Assessing the Potential of Stem Cells to Regenerate Stromal Tissue. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1425.

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

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Abstract

Purpose : Mesenchymal stem cells from human corneal stroma (CSSC) promote regeneration of transparent stromal tissue in healing corneas; however, CSSC isolated from different donors vary in their ability to induce corneal regeneration. Differentiation of RAW 264.7 macrophages induced by RANKL can be suppressed by a variety of anti-inflammatory agents. This study tested the hypothesis that suppression of macrophage differentiation by CSSC correlates with the ability to resolve stromal scarring.

Methods : CSSC isolated from limbal stroma of human corneal rims were applied to wounded mouse cornea in fibrin gel as previously described (PMID: 25504883). Corneal scarring was scored in masked fashion 14 d after wounding. Fibrotic gene expression in wounded corneas was assessed by qPCR. Differentiation of RAW 264.7 cells was induced by RANKL and assayed by tartrate-resistant alkaline phosphatase (TRAP) and qPCR for Mmp9, Acp5, and Ctsk. RNA expression patterns in six subconfluent CSSC lines were queried by RNAseq with 30M reads. Statistical significance was determined with t-tests and regression analysis with p<0.05 as a criterion.

Results : CSSC co-culture and CSSC conditioned media suppressed RANKL-induced gene expression and TRAP enzyme activity in a concentration-dependent manner (p<0.05, n=6). Conditioned media from 20 different cell lines revealed a gradient of suppression (p<0.0001, n=20). Three lines with strong suppression of differentiation (SSD) and three with weak suppression (WSD) were characterized by RNA expression profiling identifying >10 genes significantly upregulated in the SSD lines, which have not been previously identified as stem cell markers. Cell therapy using CSSC from two SSD lines suppressed scarring and fibrotic gene expression in healing mouse corneal wounds, whereas cells from two WSD lines were not effective in preventing scarring at the same concentration (p<0.001, n=8).

Conclusions : Cell lines that that effectively suppress RAW cell differentiation are more effective at promoting corneal regeneration than those with weak suppressive function. These results suggest CSSC regenerative potential is directly related to anti-inflammatory properties of the cells lines. RAW 264.7 cells can provide a standardizable assay system for identifying CSSC cells with high regenerative potential. This assay may be useful as a quality control measure in developing a cell-based reagent for human corneal therapy.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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