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Conor Ramsden, Michael Powner, Peter Coffey; Generation of Retinal Precursors using Human Embryonic and Induced Pluripotent Stem Cells: Comparison of the Efficiency of Differing Cell Culture Conditions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2218.
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© ARVO (1962-2015); The Authors (2016-present)
Pathology of the retina is responsible for the majority of cases of irreversible visual impairment worldwide. Degenerative diseases such as age related macular degeneration and hereditary diseases such as retinitis pigmentosa account for the majority of cases of retinal disease. Recent advances in cell culture techniques report the ability to generate multilayered tissues with retinal morphology that express retinal marker proteins from stem cell embryoid bodies (EBs). This will allow the fabrication of in vitro models of disease of hereditary disorders from induced pluripotent stem cells (iPS) and hopefully the generation of new tissue for transplant in degenerative disease from human embryonic stem cells (HESC). Here we describe a comparison of culture conditions with the intention of finding the most efficient method for retinal tissue generation.
3 HESC lines and one iPS line were grown on mouse embryonic fibroblasts and matrigel. Each was used to make embryoid bodies of 2000 and 500 cells. Each EB was grown in Glascows modified Eagles medium with 20% knock out serum, sodium pyruvate, antibiotics, non essential amino acids, mercaptoethanol, ROCK inhibitor and matrigel. culture conditions were compared with the addition of fetal bovine serum (FBS), wnt antagonist, and insulin like growth factor (IGF1).
Cells grown at the 2000 cell size in FBS have an increased expression of PAX6, SOX2 & RAX by immunohistochemistry. However they have a more disorganised morphology. Cells grown in the wnt angonist have a more organised morphology but do not grow at the same rate as cells without.
The addition of FBS to embryoid bodies facilitates the expression of anterior neural and retinal precursors.
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