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Vladimir Khristov, Juliet Hartford, Qin Wan, Mostafa Reza Lotfi, Kiyoharu Miyagishima, Nathan Hotaling, Arvydas Maminishkis, Juan Amaral, Sheldon S Miller, Kapil Bharti; Developing iPS Cell Derived RPE Tissue for Clinical Application. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1834.
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Age-related macular degeneration (AMD) is a leading cause of vision loss in the United States. The disease is thought to originate by a malfunctioning retinal pigment epithelium (RPE), which leads to photoreceptor cell death and vision loss. Previous work suggests replacing diseased RPE with a healthy autologous RPE sheet can provide vision rescue for AMD patients. Here we use a tissue-engineering approach combined with induced pluripotent stem (iPS) cell technology to develop an autologous RPE sheet on an artificial biodegradable scaffold.
iPS cells are differentiated into RPE using a directed-differentiation protocol that generates RPE in three phases: neuroectoderm/RPE progenitors, committed RPE, and immature RPE. RPE cells at the immature stage are purified and seeded onto biodegradable electrospun bi-layered poly lacto-co-glycolic acid (PLGA) based scaffolds. PLGA fibers with different PLGA molecular weight are electrospun in different orientations to generate scaffolds with optimized biodegradability and tensile strength. RPE tissue is validated using electron microscopy, immunostaining, gene expression analysis, fluid transport assays, and electrophysiological analysis. RPE tissue is tested in pre-clinical animals for safety and efficacy.
PLGA fiber electrospun in aligned orientation significantly increase scaffold tensile strength as compared to fibers that are electrospun in random orientation. Scaffolds with different tensile strength do not affect cell growth but affect tissue transplantation in the sub-retinal space of animal models. Electrophysiological experiments show that RPE cells on PLGA develop transepithelial resistance of more than several hundred ohms/cm2, suggesting tight electrical contacts between neighboring cells. Gene expression, immunostaining, and electron microscopy analysis show that iPS cell-derived RPE cells resemble native RPE in their molecular and structural properties. RPE cells on PLGA scaffolds also have the ability to phagocytose photoreceptor outer segments and show ability to transport water from apical to basal sides.
This work provides a fully-authenticated iPS cell derived RPE tissue for transplantation in pre-clinical animal models. This tissue behaves similarly to native tissue in its molecular and functional properties. This work also provides a protocol to develop a clinical-grade RPE tissue for transplantation in AMD patients.
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