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P. Gu, M. Wylie, L.J. Harwood, W.J. Curry, T. Cogliati; Isolation and Characterization of Retinal Stem Cells From Pig Ciliary Body . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5741.
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Retinal stem cells (RSCs) that have the capacity to proliferate in vitro and the potential to differentiate into neural retina cell types have been identified in the adult mouse and human eye. This study aims to identify RSCs from porcine ocular tissues and to investigate their capacity for self–renewal and differentiation in vitro.
Pigmented and nonpigmented epithelium of ciliary body as well as iris were surgically removed from porcine eyes. After enzymatic digestion, dissociated single cells were counted and cultured in different growth media (DMEM/F12 with or without EGF, bFGF, EGF+bFGF). Long–term self–renewal was assessed using both sphere and monolayer cultures. Cell proliferation was evaluated by cell/sphere counts and BrdU incorporation assays at each passage. Differentiation of RSCs was induced by withdrawal of growth factors and addition of serum. Immunocytochemical analysis and RT–PCR were employed to assess the nature of differentiated cells.
A small fraction (0.2–0.5%) of cells isolated from the pigmented epithelium of pig ciliary body proliferated to form clusters of cells resembling spheres. These spheres were formed in serum–free medium and contained a majority of nestin– and Pax6–immunoreactive cells. The inclusion of EGF and bFGF in the medium increased the number and size of spheres. The spheres could be passaged up to 10 times, however their proliferation potential and size decreased with passage. In monolayer culture, a 1×107–fold expansion in cell number could be obtained over two months. In both culture conditions, cells maintained the ability to give rise to neurons and glia as confirmed by their morphology and by the expression of ß–tubulin III and GFAP respectively.
RSCs are present in the pigmented ciliary body of the pig eye. They can be isolated and expanded in culture and can be differentiated into neural lineages in vitro.
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