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Danhong Zhu, Sherry T. Hikita, Xuemei Deng, Bo Lu, Lincoln V. Johnson, Dennis O. Clegg, Yuchong Tai, Ashish Ahuja, Mark Humayun, David R. Hinton; Culture of Polarized Embryonic Stem Cell-Derived RPE on Synthetic Substrates. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3191.
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RPE cell loss in the macular region is a key pathologic alteration in atrophic age-related macular degeneration (AMD). The restoration of lost RPE through transplantation of a polarized RPE monolayer is a promising new treatment for AMD. RPE cells derived from human embryonic stem cells (hESC-RPE) are a potentially unlimited resource for this therapy. This study tested two synthetic substrates, parylene (non-degradable) and poly(lactic-co-glycolic acid) (PLGA) (bio-degradable) as supports for the growth of polarized hESC-RPE monolayers for potential use in sub-retinal transplantation.
1). hESC-RPE were derived from H9 human embryonic stem cells.2). hESC-RPE were seeded on CellStart-coated (Invitrogen) substrate membranes at 105 cells/cm2 and cultured in XVIVO-10medium (Lonza) for 4 weeks.3). Scanning and transmission electron microscopy were used for ultrastructural analysis of cells and substrates.4).Whole human genome microarray analysis (Agilent) was performed on a parallel culture of hESC-RPE cells, where the cells were seeded on CellStart-coated tissue culture plates and grown in XVIVO-10 medium. RNA was collected at 4 weeks post-seeding.
hESC-RPE attached, proliferated and differentiated into polarized monolayers on both substrates. They exhibited hexagonal shape, melaninization, and apical microvilli. Microarray results showed that hESC-RPE cells grown in XVIVO10 media expressed signature RPE genes and had higher amounts of PEDF, bestrophin and RDH5, compared with controls.
Both synthetic substrates supported hESC-RPE cell growth and polarization. They are candidate platforms for differentiation and surgical manipulation of hESC-RPE monolayers for sub-retinal transplantation.
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