Characterization and in vitro proliferation of limbal stem cells a new dye for identification of ocular surface stem cells: Mitotracker deep red

In recent years, flow cytometric techniques are used more widely for isolating limbal and conjunctival epithelial stem cells. Hoechst 33342 is one of the dye for this purpose. Limbal and conjunctival epithelial stem cells transport out (efflux) the DNA binding dye Hoechst 33342 by using their G2 subtype Adenosine triphosphate Binding Cassette transporter (ABCG2) and are isolated by flow cytometry as side population (SP) cells. Transport prevents the dye from accumulating in the cell and intercalating with DNA. Unfortunately, SP sorting requires prolonged incubation with Hoechst 33342 and excitation with a UV laser, features that may lead to DNA damage.We have now identified a new ABCG2 substratum, Mitotracker deep red (MTDR) that can be used for SP cell isolation, instead of Hoechst 33342. MTDR is excited at 633 nm, accumulates in the mitochondria, not DNA, it?s application requires very short incubation and allows the collection of an expanded SP. These properties will prevent genetic material damage in clinical uses of stem cells in the future.Consistent with the stem cell phenotype, these low Mitotracker deep red dye staining (MTDR low ) cells incorporate a high proportion of cells in the quiescent, G0 state. Clonal studies on feeder 3T3 cells demonstrate that the clonal efficiency of these cells, essential to initiate to growth limbal stem cells for ocular reconstruction is affected by their cell cycle status. Understanding intracellular and extracellular control mechanisms of ocular surface stem cells will potencially improves the usage of these cells for ocular reconstruction.