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Allison E Songstad, Luke A Wiley, Cathryn Cranston, Miles Flamme-Wiese, Jeremy Hoffmann, Christine Haas, Edwin M Stone, Robert F Mullins, Budd Tucker; Generation of induced pluripotent stem cell-derived choroidal endothelial cells for the treatment of AMD. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4200.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, involves the dysfunction and eventual death of choroidal endothelial, retinal pigment epithelial (RPE), and photoreceptor cells. Induced pluripotent stem cell (iPSC)-based strategies designed to replace both photoreceptor and RPE cells are currently a major scientific focus. For some patients, success of these approaches may also require replacement of choroidal endothelial cells (ECs). The purpose of this study was to generate choroidal ECs from two Tie2 GFP iPSC reporter lines to develop efficient differentiation and transplantation protocols.
Dermal fibroblasts from the Tie2 GFP mouse (carrying a GFP reporter gene under the control of the EC-specific Tie2 promoter) were isolated and reprogrammed into two iPSC lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. iPSC potency was characterized via RT-PCR, immunocytochemistry (ICC), and teratoma formation assays. Tie2 GFP iPSCs were differentiated into ECs using a co-culture method with either the RF6A choroidal EC line or primary mouse choroidal ECs. The Tie2 GFP iPSC-ECs from both lines were characterized via RT-PCR and ICC for various EC-specific and choroidal EC-specific markers.
Both iPSC lines were subjected to co-culture with either the RF6A cell line or primary mouse choroidal ECs and differentiated into vascular ECs. The two Tie2-GFP iPSC-derived EC lines expressed the EC markers CD31, CD34, Tie2, VE-Cadherin, and VWF, as determined by RT-PCR, expressed the choroidal EC-specific markers ICAM1, as determined by RT-PCR, and carbonic anhydrase IV, as determined by RT-PCR and ICC, and were morphologically indistinguishable from native choroidal ECs that expressed ZO-1 and VE-Cadherin in cell-cell adhesions.
We have successfully generated Tie2 GFP iPSCs that can be efficiently differentiated into choroidal-like ECs. This work has paved the way for future studies focused on investigating AMD pathophysiology and EC replacement.
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