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Danhong Zhu, Dennis O. Clegg, David R. Hinton; Polarized Human Embryonic Stem Cell-Derived RPE Maintains its Monolayer Integrity and Function after Long-term in vitro Culture. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5924.
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Retinal pigment epithelial (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. However, knowledge is lacking about the optimal time-range of hESC-RPE culture in vitro, and how that will affect the long term survival of grafted RPE monolayer in vivo. This study investigated the morphology, gene profile and function of the long term in vitro cultured hESC-RPE.
1). hESC-RPE was derived from H9 human embryonic stem cells. 2). hESC-RPE were seeded on matrigel (BD biosciences) coated culture plates at a density of 105 cells/cm2 and cultured in XVIVO-10 medium (Lonza) for 1, 2 and 4 months. 3). ELISA, qRT-PCR and immunofluorescent staining were used to detect the expression and secretion of RPE cell signature genes and proteins.4). Scanning and transmission electron microscopy were used for ultrastructural analysis of cell structures.
hESC-RPE attached, proliferated and differentiated into polarized monolayers on matrigel coated plates within 1 month. The cells kept their hexagonal shape, melaninization, and apical microvilli after long term culture. The secretion levels of pigment epithelial-derived factor (PEDF) and vascular endothelial growth factor (VEGF) did not decrease in 4 month culture by ELISA. The expression level of RPE signature genes had no significant difference among the different times the cells were cultured.
After forming the polarized monolayer, hES-RPE can survive for long periods of time in vitro. The polarized RPE not only kept their specific morphology, but also expressed high levels of RPE marker genes in long term cultures. The finding provides the basis for the in vitro culture time range before transplantation and indirect evidence that polarized RPE cell sheets could havelong term survivalin the sub-retinal space after transplantation, which is a necessary requirement for cell replacement therapy for AMD.
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