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N.-K. Wang, M. Kasanuki, R. J. Davis, J. Chou, K. Janisch, J. Tosi, C.-S. Lin, S. Tsang; Functional Analysis of Mouse Embryonic Stem Cells-Derived Retinal Pigment Epithelium Cells Transplantation in Rpe65rd12/Rpe65rd12 Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4537. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine if mouse Embryonic Stem Cell (mESC) can differentiate into RPE cells in vitro and if these cells can rescue retinal function in the mouse retinal degeneration model Rpe65rd12/Rpe65rd12.
To induce stem cell differentiation into RPE, mESCs were cultured on PA6 cells with media containing bFGF, dexamethason, and cholera toxin. mESCs were cultured for six days and observed for morphological changes, such as pigmentation and epithelial-like shape. In addition, these cells were assessed for expression of known RPE specific markers (RPE65 and Bestrophin) by immunocytochemistry staining. The specificity of anti-RPE65, and anti-bestrophin antibodies were tested on the human RPE cell line (ARPE-19), using immunocytochemistry and western blotting. To assess the function of PA6-treated mESCs cells in vitro, 2 x 106 cells were injected into the subretinal space of postnatal day 5 (P5) Rpe65rd12/Rpe65rd12 mice. Frozen retinal sections were analyzed to determine the location and morphology of transplanted cells. Finally, electroretinogram (ERG) was performed on injected mice to evaluate the functional outcome one, two, and four months after injection.
After six days culturing on PA6 cells, mESCs showed signs of morphological change, but no pigment expression was detected. Observations of any additional morphological changes, after long-term culturing, will be presented. However, anti-RPE65 and anti-bestrophin antibodies stained PA6-treated mESCs and ARPE-19 cells, while secondary antibodies alone failed to stain. Frozen retinal sections showed transplanted cells were in the subretinal space 10 days after injection. Data from serial ERGs experiments are in progress and will be presented.
Our data indicate that PA6-treated mESCs express RPE65 and bestrophin, suggests that mESCs have the potential to differentiate morphologically and functionally to RPE. If retinal function can be rescued, the use of stem cell-based therapy may represent a future option in treating some forms of retinal degeneration. Otherwise, further studies are required to identify optimal conditions for generating functional RPE cells from mESC, such as improved cell lines, culture conditions, and transplantation techniques.
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