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Allison E Songstad, Luke A Wiley, Erin R Burnight, S Scott Whitmore, Emily E Kaalberg, Megan J Riker, Edwin M Stone, Robert F Mullins, Budd A Tucker; Development of a step-wise protocol for the generation of human induced pluripotent stem cell-derived choroidal endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1136.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) involves dysfunction of choroidal endothelial cells (CEC), retinal pigment epithelial cells (RPE), and photoreceptor cells (PRs). Human induced pluripotent stem cell (iPSC)-based strategies to replace PRs and RPE are a major scientific focus. However, success of these approaches may require the replacement of damaged and lost CECs, which degenerate early in the pathogenesis of AMD. The purpose of this study is to design a step-wise differentiation protocol to generate iPSC-derived CECs.
Fibroblasts from a patient with normal ocular history were isolated from a skin biopsy and reprogrammed into iPSCs via transduction with a non-integrating Sendai virus driving expression of OCT4, SOX2, KLF4, and c-MYC. Pluripotency was assessed via TaqMan Scorecard Assay and immunocytochemistry (ICC). RNA sequencing analysis of cultured monkey RF/6A CECs and 2-D gel/mass spec analysis of RF/6A-conditioned medium identified seven secreted proteins known to be involved in vascular development. Using the Taguchi statistical strategy, media were developed using combinations of the seven proteins to drive CEC differentiation. In a separate strategy, each protein was eliminated one at a time from the differentiation protocol to identify those that were crucial for CEC differentiation. The iPSC-CECs from both methods were characterized via rt-PCR and ICC for EC- and CEC-specific markers.
Using each approach, patient-specific iPSCs cultured in media containing CTGF, TWEAKR, and VEGF-B differentiated into CECs that expressed the EC-specific markers CD31, CD34, eNOS, FOXA2, ICAM1, TIE2, and VE-Cadherin, and the CEC-restricted markers CA4 and TTR. CTGF is required for CEC differentiation, whereas TWEAKR and VEGF-B enhanced CEC differentiation. CTNNB1, PDPN, RTN4, and SHC1 did not play a major role in CEC differentiation. The iPSC-CECs fed CTGF, TWEAKR, and VEGF-B formed vascular tube networks morphologically identical to those formed in primary human CEC cultures.
We describe two strategies used to develop a stepwise differentiation protocol for the derivation of CECs from patient-specific iPSCs. In addition to being useful for cell replacement, patient-specific CECs, in combination with RPE and PRs, will be invaluable to accurately interrogate the pathophysiology of AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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