Sphere Initiating Cells in the Mouse Corneal Stroma Exhibit Multipotency in Addition to the Keratocyte Phenotype in vitro

Abstract

Abstract: : Purpose: To analyze the self–renewal capacity and multipotency of sphere initiating cells in the mouse cornea. Methods: Dissociated mouse corneal stromal cells were cultured in serum–free DMEM/F12 medium supplemented with EGF and bFGF to initiate spheres. Methylcellulose culture method was used to assess self–renewality of sphere–forming cells, which were analyzed by the Hoechst 33342 dye exclusion assay to isolate cells with side–population (SP) phenotype. Cells from P6 to P10 spheres were subcultured onto plastic dishes or type I collagen gels for phenotype analysis. Expression of keratocan, aldh, CD34, cytokeratin K12 (K12), Pax6, and musashi–1 was analyzed by RT–PCR. Immunocytochemistry was done against sphere cells subcultured in condition medium to express α–SMA (myofibroblast), ßIII–tublin and MAP2,(neuron), and O4 (glia cell). Oil–red staining was done to identify lipid producing adipocytes. Results: Primary keratocytes formed spheres, which were cultured for over 12 passages. A high fraction of these cells demonstrated the SP phenotype by FACS. Subculture using a methylcellulose sustrate showed regeneration of spheres from single cells. Suspended sphere cells expressed vimentin, keratocan, CD34 and lumican, but were negative for K12 and Pax6. Sphere cells subcultured on plastic showed dendritic morphology characteristic of keratocytes, and maintained keratocan, Aldh and CD34 expression in serum–free medium. Sphere cells subcultured with serum became fibroblastic, and expressed α–SMA when stimulated by TGF–ß. The neural progenitor marker musashi–1 was detected in spheres, and dissociated sphere cells subcultured in condition medium up–regulated the neural and glial markers ßIII–tublin, MAP2, O4, and GFAP. Lipid producing adipocytes were also observed by oil–red staining. Conclusions: Stromal spheres cells contain self–renewing keratocyte progenitors with intact keratocyte phenotype and the ability to differentiate into neurons, glial cells and adipocytes.

Keywords: cornea: stroma and keratocytes • cornea: basic science • gene/expression