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J. Yang, P. Gu, H. Klassen; Molecular Characterization of Human Retinal Progenitor Cells During Long Term Culture. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2649.
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Human retinal progenitor cells (hRPC) are a potential therapeutic agent in the setting of retinal degeneration, however, supplies of these cells are restricted by a limited capacity for growth in culture. We sought to extend the limits of hRPC growth and provide a characterization of heavily passaged hRPCs, including karyotpe analysis and molecular evidence of phenotypic potential.
Neural retinal tissue of 18 weeks GA was minced and enzymatically digested. Cells were seeded into proliferation medium containing either DMEM:F12- or Ultraculture-based medium, supplemented with N2, Glutamax, EGF (20 ng/ml) and bFGF (20 ng/ml). Cells were passaged at 80-90% confluence every 4-6 days, continuously, for 4 months. Total RNA was isolated on days 17, 22, 27, 38, 48, 72 and 99, and subjected to quantitative PCR analysis. At passage 6, cells were fixed with 4% PFA and immunolabeling performed. Karyotype and FISH were performed at passage 19.
Human RPCs were still proliferating after 4 months of continuous culture in DMEM:F12-based medium. Expression of markers associated with progenitors (nestin, vimentin, Ki-67, CD9, CD81, FUT4) as well as lineage (β3-tubulin, PKC-α), immune function (β2-microglobulin, HLA-DPa1), development (c-myc, KLF4), apoptosis (caspase1, caspase3, annexinV) and GDNF were up-regulated at progressive time points. In contrast, Sox2, Six6, Notch1, Map2, recoverin, and GFAP were down-regulated. Compared to human fibroblasts, Sox2 and Map2 were much more highly expressed by hRPCs. Immunolabeling showed more than 95% of cells to be positive for progenitor cell markers (nestin, vimentin), 50% for the proliferation marker Ki-67, and 5% for GFAP. In addition, karyotype and FISH showed 86% of cells to be normal and 14% tetraploid (8% aneuploid considered upper limit of normal) at passage 19.
hRPCs derived from the fetal retina can be expanded extensively, although not indefinitely, in vitro. After long term growth in culture, the majority of cells continue to express progenitor markers and retain a normal karyotype, as well as the ability to express markers of neuronal and glial lineage in vitro.
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