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Yiqin Du, Martha L. Funderburgh, Hongmin Yun, Joel S. Schuman; Survival of Trabecular Meshwork Stem Cells in the Presence of Glaucoma-Associated Factors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3241.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we identified a cell population from human trabecular meshwork with properties of multipotent stem cells. These trabecular meshwork stem cells (TMSC) home to mouse trabecular meshwork (TM) in vivo adapting a TM phenotype. This study tests the hypothesis that TMSC resist stimulation by soluble factors associated with glaucoma better than TM cells.
DiO-labeled TMSC were expanded two passages, stained with Hoechst dye and stem cell markers ABCG2, Notch1, MUC1 and TM marker CHI3L1. Passaged TMSC and TM cells were stimulated with glaucoma-associated factors dexamethasone, TGFβ2 or TNFα. Apoptosis was detected by Annexin V staining and flow cytometry. qRT-PCT, immunoblotting, and immunofluorescent staining compared expression of myocilin, ELAM1, NF-ΚB, AQP1 and CHI3L1. TMSC, TM cells, and fibroblasts were transplanted into mouse anterior chambers for 4 weeks and 4 months. Viability of the injected cells was assessed using Calcein AM staining. Immunofluorescence examined inflammatory cells (CD45) and human-specific MUC1 and CHI3L1.
Expanded TMSC cultures had heterogeneous DiO staining indicating cells with different rates of replication. DiO-bright cells had reduced Hoechst dye staining consistent with dye-efflux typical of stem cells. These cells stained for stem cell markers ABCG2, Notch1, and MUC1. DiO-dim, rapidly dividing cells, expressed TM marker CHI3L1. Stimulation with dexamethasone, TGFβ2 or TNFα induced expression of glaucoma markers myocilin, ELAM1, and NF-ΚB but reduced AQP1, CHI3L1 in both TMSC and TM cells. Changes in TMSC were quantitatively less than those of TM cells. After transplantation into murine anterior chamber, no CD45 positive cells were seen in the TM of mice with TMSC. Injected TMSC remained viable 4 months after injection and expressed either MUC1 or CHI3L1. Fewer injected fibroblasts colonized the TM region. Apoptosis of injected fibroblasts was greater than that of injected TM or TMSC.
The slow-cycling and asymmetric replication of TMSC suggests a role in replacement of lost or damaged TM cells in vivo. The observation that TMSC tolerate stimulation with glaucoma-associated factors in vitro and survive in vivo for up to 4 months, supports the hypothesis that TMSC provide a source of healthy TM cells in vivo.
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