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Martha L Funderburgh, Golnar Shojaati, Mary Mann, Yiqin Du, James L Funderburgh; Controlling the Regenerative Potential of Corneal Stromal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):905.
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© ARVO (1962-2015); The Authors (2016-present)
Mesenchymal stem cells from human corneal stroma (CSSC) induce regeneration of transparent stromal tissue during wound repair in mice and are currently in process for usage in clinical trials for therapy of existing stromal scars. The mechanism by which corneal fibrosis is prevented, however, is not fully understood. In the absence of adhesive substratum, CSSC associate into spheroids. In an effort to understand factors controlling CSSC regenerative potential, this study compared properties of sphere-derived CSSC (Sp-CSSC) with substrate-attached CSSC (At-CSSC).
Limbal stromal tissue of donor corneal rims was dissected and collagenase digested, and CSSC were expanded at clonal density as described (PMID: 25504883). Spheres from passage 3-4 CSSC were formed in polyhema-coated dishes or in polypropylene tubes in DME/F12, B27, FGF2, and EGF at 105 cells/ml for 3 days, then dissociated with TrypLE to yield Sp-CSSC. Gene expression was examined by qPCR. Suppression of scarring was examined in a mouse model of corneal wound healing with 2x104 CSSC in a fibrin gel applied at the time of wounding. Neutrophil infiltration was assessed by ELISA for myeloperoxidase at 44 hr after wounding. Statistical significance was determined with t-test analysis of replicates using p<0.05 as a criterion.
Sp-CSSC had marked upregulation of genes associated with immunosuppressive activity including TSG-6, IL10, and COX2 compared to At-CSSC. TGFβ3, a cytokine associated with scarless wound healing, was upregulated > 100-fold as were CXCR4 and CXCL12, proteins involved in stem cell homing. In corneal wounds, Sp-CSSC completely suppressed neutrophil infiltration. At 14 days, expression of fibrotic markers (Fap, Tnc, Acta2,Tgfb1,Col3a1, Sparc) was significantly reduced in Sp-CSSC-treated wounds at levels similar to that of unwounded tissue. Suppression of fibrosis by Sp-CSSC was not statistically different from that of At-CSSC.
Sphere formation selects a CSSC population expressing high levels of genes associated with regenerative potential. In vivo, Sp-CSSC suppressed inflammation and prevented stromal fibrosis. These results suggest that sphere formation may help standardize the regenerative potential of cell lines from different donors, and may allow use of lower cell dosages in therapeutic applications.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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