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Coralia C Luna, Megan Parker, Pratap Challa, Pedro Gonzalez; Evaluation of the potential of iPSCs as a tool to study Pseudoexfoliation Syndrome. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4911.
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© ARVO (1962-2015); The Authors (2016-present)
A major limitation to investigate the pathophysiology of pseudoexfoliation (PEX) syndrome and develop effective treatments is the lack of experimental models that recapitulate the main alterations in production and maturation of extracellular material observed in PEX patients. The objective of this research was to test the potential of induced pluripotent stem cells (iPSCs) from PEX donors to generate an in vitro model for PEX syndrome.
Primary cultures of dermal fibroblasts were generated from skin biopsies from 2 confirmed PEX donors and reprogramed into iPSCs by transfection with plasmids: Oct4+P53 inhibitor, Sox2, Klf4, L-Myc, Lin 28 and Ebna. Reprograming of selected clones was confirmed by immunofluorescence. The iPSCs were differentiated into vascular smooth muscle cells or vascular endothelial cells by incubation in specific differentiation media and differentiation levels were evaluated by western blot of SMA and SM22 for smooth muscle, and VECAD and CD31 for vascular endothelial cells. iPSCs procured from the NINDS Repository from 4 non-PEX donors were used as controls and differentiated under the same conditions. Differentiated cultures were examined by transmission electron microscopy at 7 and 14 days after reaching confluency to evaluate production of microfibrils and maturation of elastic fibers.
iPSCs from both control and PEX donors could be differentiated into smooth muscle and vascular endothelial cells with levels of expression of differentiation markers that varied among the cell lines particularly for vascular endothelial cells. Both cell types showed extensive production of basement membrane components including microfibrils and elastic fibers. In absence of any additional stressors or regulatory factors, cells form PEX donors showed more microfibrils than controls and, although formed thicker electron dense bundles, did not mature into normal elastic fibers suggesting differences in elastogenesis between cells derived from PEX donors and controls.
The observed extensive production of basal membrane components and differences in elastogenesis between PEX and control cells suggest that differentiation of iPSCs from PEX donors into cell types capable of forming basement membranes, such as smooth muscle and vascular endothelial cells, can be a useful approach to develop an in vitro experimental model to investigate the molecular pathogenesis of PEX syndrome.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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