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P. Gu, J. Yang, Y. S. Lee, K. Narfstrom, I. K. Kong, H. Klassen; Isolation, Proliferation, and Characterization of Retinal Progenitor Cells From Domestic and RFP-Transgenic Cats. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5137.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal progenitor cells (RPCs) with the capacity to proliferate in vitro and the potential to differentiate into retinal cell types in vivo have been isolated from the developing rodent, pig and human. Hitherto, this has not been achieved in the cat, among the most studied models of visual function. Here we isolate RPCs from the cat retina and evaluate expression of a range of transcripts at different time points in culture.
Neural retinas were surgically removed from donor domestic or transgenic fetuses at 45 days gestational age (GA) and enzymatically digested. Cells were seeded in proliferation medium containing EGF (20 ng/ml) and bFGF (20 ng/ml) which was changed every 2-3 days. Cells were passaged at 80-90% confluence and total RNA isolated at days 13, 31, 52, 97 and 114 followed by quantitative PCR analysis. Passage 18 (day 104) RPCs were grown on 4-well chamber slides coated with fibronectin, either in proliferation medium or differentiation medium with 10% fetal bovine serum (FBS) in place of mitogens and medium changed every 2-3 days. Day 7 after plating, cells were fixed with 4% paraformaldehyde and immunolabelling performed.
Feline RPCs were still proliferating at 4 months of continuous culture. The expression of progenitor markers (nestin, Sox2, vimentin, Pax6) as well as lineage markers (DCX, β3-tubulin, Map2, recoverin, PKC- and GFAP) were down-regulated from day 13 to day 52, after which expression was maintained at a stable lower level. Immunolabelling showed more then 80% of cells positive for progenitor cell markers (nestin, vimentin); in differentiation medium, a large number of cells were positive for β3-tubulin and rhodopsin, and a few for GFAP. In addition, RFP-transgenic cells exhibited upregulation of red fluorescent protein in culture, with a few cells positive on day 0 but over 90% positive by day 6.
RPCs can be derived from the fetal cat retina and expanded extensively in vitro. After long term culture, the majority of cells continue to express progenitor markers and retain the ability to differentiate along neuronal and glial lineages in vitro. The RFP-transgenic RPCs represent an attractive donor cell population for allogeneic transplantation studies in cat models of retinal disease.
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