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K V Chalam, Sankarathi Balaiya, William Scott, Lee Ronald Ferguson; Spontaneously Differentiated Human Embryonic Stem Cell derived Retinal Pigment Epithelium Cells express RPE-65 and ZO-1 proteins. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1362.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the protein expression profile of the retinal pigment epithelium-65 (RPE65) and zonular occludins-1 (ZO-1) markers during spontaneous maturation of precursor cells into human embryonic stem cell derived RPE (hESC-RPE)
WA09-DL-11 feeder dependent human embryonic stem cells (hESC) were seeded onto a confluent layer of Mitomycin C inactivated mouse embryonic fibroblasts. hESC growth and differentiation was monitored for the presence of pigmented embryoid body (EB) development. Pigmented EB clumps were isolated following dissection from hESC colonies. EBs were seeded onto 6-well gelatin coated plates and allowed to form an expansive monolayer of hESC-RPE cells. Cells were then passaged 2 - 3 weeks after plating EB. Following passage cells were re-plated onto four gelatin coated wells. Each well represented different time points of extraction: 5, 13, 21, and 28 days post passage. Cells were lysed and then centrifuged to acquire supernatant proteinaceous material. Cellular lysate supernatant was then quantified and subjected to western blot analysis. Antibodies to RPE65 and ZO-1 were used for the analysis. Following western blot procedure densitometry was performed to quantify protein expression at various time points.
hESC-RPE post passage day 5 densitometry value was 1782.841 optical density units (odu) for RPE65 and 295.021 odu for Zo-1. On day 13 there was a 1.4 fold increase for RPE65 (2556.024 odu) and 16.5 fold change in expression for ZO-1 (4856.589 odu). Protein expression continued to rise for RPE65 and ZO-1, as day 21 levels showed a 1.6-fold (2832.953 odu) and 20.5-fold (6052.439 odu) increase, respectively, when contrasted to day 5 values. Finally on day 28, expression profiles plateaued as RPE65 levels only showed a 1.2-fold increase from baseline (2303.79 odu), while ZO-1 demonstrated a 20.4-fold increase from baseline (6019.196 odu).
Precursor marker characterization of hESC-RPE demonstrates RPE-65 and ZO-1 expression as early as 5 days with a plateau apparent at day 28. Further investigation into days prior to day 5 and after day 28 are needed for precise delineation of onset of expression for these two markers. This study demonstrates the pattern of RPE65 and ZO-1 expression, which provides further insight into the proper characterization of hESC-RPE cells as true RPE cells.
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