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Lee Ferguson, Sandeep Grover, Kakarla Chalam; Evaluation of Spontaneous Differentiation of hESCs into RPE Cells in the Absence of bFGF. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2211. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate human embryonic stem cell (hESC) differentiation into retinal pigment epithelial like cells (hESC-RPE) following basic fibroblast growth factor (bFGF) removal. This study characterizes the morphology of hESC-RPE precursor cells during this spontaneous transformative process.
The WA09-DL-11 feeder dependent stem cell line were thawed from liquid nitrogen and seeded onto a gelatin coated well with a confluent layer of Mitomycin C inactivated mouse embryonic fibroblasts. hESCs were cultured in a growth media containing bFGF with media exchanges occurring daily. After 2 weeks, hESC growth media was replaced with a nutrient media devoid of bFGF for embryoid body (EB) development. Pigmented areas were dissected from hESC colonies and isolated into low attachment wells. EB were incubated for 2 weeks with media replacement occurring every other day. EB were then removed from suspension and seeded onto a gelatin coated well and allowed to expand in RPE expansion. Precursor stage characterization via light microscopy was performed following EB sub-culturing on to gelatin coated wells.
Day one after EB sub-culturing, round globular cells with scant clear cytoplasms encircling pigmented granular centers were visualized. On day 3, hESC-RPE precursor cells demonstrated an ellipsoid shape with central vacuole core encompassing 2 - 4 pigmented granules. The center vacuolated area was surrounded by cytoplasm containing multiple circular pigment granules at varying degrees of density per cell. On day 11, precursor cells displayed cellular polarity with one pole containing the vacuolated center while the opposite pole possessed an abundance of pigmented granules. The cells have also changed configuration from ellipsoid to a polygonal cobblestone pattern. On day 34, the cells no longer conveyed a polarity; instead there was a central area of densely packed pigment granules. Additionally there was a translucent region around the pigmented core. At the outer extent of the cell there was a darkly pigmented lacey boarder that encapsulated the cell within the polygonal cobblestone shape.
The potential for hESCs to differentiate into characteristic RPE emphasizes the applicability of hESC-RPE for therapeutic cellular replacement. The importance in understanding the morphologic features of the hESC-RPE is essential when considering implementation to clinical trials.
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