Homeostatic renewal and regeneration of the ocular surface linings, the conjunctival, and limbo-corneal epithelia ultimately depends on a small population of stem cells that self replicate throughout life. Isolation of these stems cells in the viable state will provide new venues to use them in the treatment of ocular disease, or in fundamental studies on mechanisms of ocular development and repair. Isolation of cells in viable form is generally dependent on identification of plasma membrane surface epitopes to 'mark' these cells without damaging or modifying them for isolation by sorting or panning. A major impediment to isolation of ocular surface stem cells is the absence of known stem cell markers or properties useful for their recognition and isolation. In this context, recent experimental evidence suggests that many somatic stem cells may share a common detoxification mechanisms underpinned by over-expression of a particular multidrug resistance transporter, known as ABCG2/BCRP/MTX. When ABCG2-rich cells are incubated with the DNA binding dye Hoechst-33343 (Ho333) and analyzed by fluorescence activated cell sorting (FACS) using a UV excitation source, highly efficient Ho333-efflux results in a fluorescence spectral shift. This shift causes cells to appear in emission plots as a characteristically isolated group of cells to the left (blue) side of the normal blue/red ratio. We have now confirmed the presence of this protein in the ocular surface epithelia and have established effective ways to isolate SP cells. These cells comply with the main known or expected features of the epithelial stem cell phenotype including, a) their remarkable lack of intracellular 'granularity' and a small size; b) unique ability to initiate clonogenic proliferation following prolonged exposure to phorbol ester; c) slow recruitment to proliferation when set in culture. The objective of this application is to exploit the possibility of isolating sufficient amounts of these cells from the ocular of farm mammals to allow the identification of the family of genes that are differentially expressed in the ocular surface stem cells, as well as those genes whose expression hallmark the start of differentiation. Given our current very limited understanding of stem cells, acquisition of a gene library is an essential, high impact step in the long path to unravel the process underlying the functionality of these privileged cells.
