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Allison E Songstad, Cathyrn M Cranston, Miles J Flamme-Wiese, Edwin M Stone, Robert Mullins, Budd A Tucker; Generation of iPSC derived vascular endothelial cells for the treatment of AMD.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3451.
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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, is characterized by the dysfunction and eventual death of choroidal endothelial, retinal pigment epithelial (RPE), and photoreceptor cells. Stem-cell based treatment strategies designed to replace both photoreceptor and RPE cells are currently a major scientific focus, and success of these approaches will undoubtedly also require replacement of choroidal vasculature. The purpose of this study was to generate a Tie2-GFP iPSC reporter line to develop efficient vascular endothelial cell differentiation and transplantation protocols.
Dermal fibroblasts from the Tie2-GFP mouse (carrying a GFP reporter gene under the control of the endothelial cell-specific Tie2 promoter) were isolated and reprogrammed into induced pluripotent stem cells (iPSCs) via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. iPSC potency was characterized via RT-PCR, immunocytochemistry, western blotting and teratoma assays. Tie2-GFP iPSCs were differentiated into embryoid bodies using both a co-culture method as well as a modified version of our previously developed step-wise differentiation protocol. Translatability of developed protocols was tested using human control iPSCs.
Tie2-GFP iPSCs, generated from murine fibroblasts, expressed the pluripotency markers Nanog, Oct4, Sox2, Klf4, and c-Myc as determined by RT-PCR, western blot analysis and immunocytochemistry. Following both embryoid body formation and transplantation into immune compromised SCID mice, undifferentiated cells formed tissues specific to each of the 3 embryonic germ layers. Pluripotent iPSCs subjected to co-culture with the monkey choroidal endothelial cell line RF/6A differentiated into vascular endothelial cells that expressed the choroidal endothelial markers VE-cadherin and CD34 and were morphologically indistinguishable from native choroidal endothelial cells.
We have successfully generated Tie2-GFP iPSCs and used them to develop vascular endothelial cell differentiation protocols. This work has set the stage for future studies focused on investigation of disease pathophysiology and endothelial cell replacement.
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