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Juliet Hartford, Helen May-Simera, Jason Silver, Janine Davis, Kiyoharu j Miyagishima, Vladimir Khristov, Omar Memon, Andrea Li, Sheldon S Miller, Kapil Bharti; Primary Cilium Regulates iPS Cell Derived RPE Maturation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):704.
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The retinal pigment epithelium (RPE) is a ciliated monolayer of cells situated adjacent to retinal photoreceptors. The RPE is critical for maintaining the health and integrity of photoreceptors. Ciliopathies are a class of disorders that affect cilia formation or functioning and lead to photoreceptor degeneration. The role of cilia proteins in photoreceptor development is well understood, however, its function in the RPE is not known. The goal of this study is to understand the role of the primary cilium in RPE development and function in mouse and human models.
RPE cells in WT and two different cilia mutant mice (Bbs8-/- and Mkks-/-) were analyzed using immunohistochemistry, scanning electron microscopy, and qPCR. Primary cilium in human induced pluripotent stem (iPS) cell derived RPE was manipulated using cilia inducers and inhibitors. Immunohistochemistry, scanning and transmission electron microscopy, gene expression, electrophysiology, intracellular calcium measurements, and fluid transport were used to determine the maturity of iPS cell derived RPE.
Ciliopathy mutant mice displayed persistent upregulation of developmental transcription factors MITF and PAX6 in the RPE. This was consistent with incomplete maturation of RPE in mutant mice. iPS cell derived RPE treated with primary cilia agonists and antagonists reinforced this result. Induction of primary cilium in iPS cell derived RPE improved melanogenesis, induction of apical processes, barrier resistance across the monolayer, and ability to transport fluid. All these properties are consistent with improved maturation of RPE cells. In contrast, suppression of cilium function dramatically reduced RPE maturation.
Disrupted cilia function results in delayed/improper RPE maturation that could contribute to retinal degeneration in ciliopathy patients. Cilium induction in iPS cell derived RPE leads to physiologically and phenotypically stable RPE cells. We propose that the primary cilium helps mature RPE cells through modulation of RPE developmental pathways and that manipulation of these pathways can help generate mature RPE cells that serve as more effective disease and cell-therapy models.
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