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Y. Du, E.C. Carlson, M.L. Funderburgh, J.L. Funderburgh; Differentiation of Human Stem Cells in Mouse Corneas After Xenotransplantation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1813.
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The mouse cornea provides an in vivo model for investigating cell–based therapy for corneal scarring. Recently we described stem cells from human corneal stroma (CSSC) which adopt characteristics of keratocytes in vitro (Du et al., Stem Cells 23:1266, 2005). In the current study we injected these cells directly into mouse corneal stroma and analyzed production of corneal stromal matrix molecules and stromal–specific genes.
Human CSSC were isolated by cell sorting using Hoechst 33342 dye exclusion. Human corneal fibroblasts cultured in fetal bovine serum served as the control. CSSC or fibroblasts pre–labeled with DiO were injected directly into the corneal stroma of normal mice. Corneal transparency and cell localization was followed by in vivo biomicroscopy. The expression of human specific keratocan was examined by immunohistology, western blotting, and qRT–PCR. Immune cell infiltration was examined by H&E histology of paraffin sections. Viability of the injected cells was examined by uptake of calcein red–orange dye and fluorescence–activated cell sorting (FACS).
Mouse corneas cleared rapidly after injection of 2 ul of cell suspension containing 50,000 cells. Uncomplicated injections elicited no detectible inflammatory response or rejection. Fibroblast–injected corneas exhibited a slight haze, but CSSC corneas remained clear. Fluorescently labeled injected CSSC spread throughout the cornea, adopted a dendritic morphology and remained visible for >8 months. 6 weeks after injection, the presence of viable injected fluorescent cells analyzed by FACS showed that approximately the same number of injected cells could be recovered with 85% viability. The injected CSSC expressed mRNA for the stromal marker keratocan and human keratocan protein was detected in the mouse stroma by western blotting and immunohistology.
Corneal stroma has long been known for its immune privilege. In these experiments we show that trans–species xenotransplantation of stem cells into the corneal stroma elicits no frank immune rejection, allowing the cells to differentiate into keratocytes, producing human stromal matrix components. This study shows the mouse to be a useful model for stromal cell–based therapy and highlights the potential for use of allogenic stem cells in stromal cell–based therapy or bioprostheses.
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