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Michael Burrow, Yiqin Du, Martha L Funderburgh, Richard Dannenberg, Mary Mann, Sayan Basu, James L Funderburgh; Mechanisms of Corneal Stromal Remodeling by Corneal Stromal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5171.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell therapy restores transparency to scarred corneas in animal models and may present an alternative to keratoplasty in humans. We hypothesize that stem cells remodel corneal stroma via a temporal program of degradation of pathological extracellular matrix (ECM) with matrix metalloproteinases (MMPs) followed by synthesis and deposition of new transparent ECM. Furthermore, we hypothesize that this process is distinct from the remodeling that takes place by fibroblasts during wound healing.
Corneal stromal stem cells (CSSC) were expanded from post-surgery human corneal rims, differentiated to keratocytes in Advanced-DMEM medium with FGF2 and ascorbate-2-phosphate on collagen substrata, and were harvested at 1, 2, 3, 5, and 7 days for RNA analysis by qPCR or NanoString nCounter. Collagen degradation was assessed by degradation of fluorescent collagen substrata after 24-48 hr in differentiation medium. MMP expression in vivo was assessed by RT-PCR analysis of CSSC recovered by FACS, 8 days after injection into mouse corneal stroma. Enzymatic activity of MMPs was assessed using fluorogenic peptide substrates.
As CSSC differentiated to keratocytes they transiently increased expression of mRNAs for MMPs 1, 3, 11, 13, and 14. Fluorimetric MMP assays confirmed secretion of active MMPs during culture. MMP action was visualized by digestion of fluorescent collagen substrate by CSSC, a process completely blocked by MMP inhibitors GM6001 and TIMP2. Inhibition of membrane-bound MMP-14 with blocking antibody or with siRNA demonstrated a key role for this MMP in ECM remodeling. MMPs were also upregulated in corneal fibroblasts under similar conditions; however, gene expression patterns of fibroblasts and CSSC differed for several MMPs and TIMPs. Preliminary in-vivo experiments confirmed upregulation of CSSC MMP mRNA as the cells differentiated in vivo.
These experiments show that differentiation of CSSC to keratocytes stimulates transient expression of a specific set of MMPs when these cells are exposed to a collagenous environment. The results support a role for specific MMPs in remodeling corneal ECM during corneal regeneration by stem cells. Together, these results support our hypothesis that stem cells employ a unique molecular program to remodel pathological tissue. Moreover, they indicate that remodeling by CSSC is distinct from remodeling by fibroblasts during wound healing.
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