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Ruchira Singh, Wei Shen, Xiangrong Guo, Enio T. Perez, David Kuai, Lynda S. Wright, Bikash Pattnaik, David M. Gamm; Functional Comparison Of RPE Cultures Expanded From Differentiated Human iPS Cells And Prenatal Eye Tissue. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5906.
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© ARVO (1962-2015); The Authors (2016-present)
Human induced pluripotent stem cells (hiPSCs) can be differentiated into retinal pigment epithelium (RPE) with varying efficiency, making hiPSCs a promising cell source for patient-specific retinal disease modeling, drug screening, and perhaps cell-based therapies. To facilitate these applications, it would be useful to isolate, passage, and expand RPE from mixed, differentiating hiPSC cultures. Therefore, we examined selected physical, molecular, and functional properties of passaged hiPSC-RPE and compared the results to those obtained from prenatal human RPE.
hiPSC lines from two different families were differentiated toward a retinal fate using our previously described protocol. Primary hiPSC-RPE clusters were dissected, dissociated, expanded, and evaluated by RT-PCR and immunocytochemistry to assess the expression of RPE-associated genes and proteins. In addition, we examined the capacity of passaged hiPSC-RPE to form tight junctions, phagocytose bovine photoreceptor outer segments, and release calcium in response to ATP stimulation, using cultured human prenatal RPE as a control
hiPSC-RPE could be passaged at least twice and expanded >103-fold. Passaged hiPSC-RPE formed monolayers with characteristic pigmentation and morphology, expressed appropriate gene and protein markers, and formed tight junctions with transepithelial resistances (742.9 ± 37.7) on par with human prenatal RPE . Moreover, passaged hiPSC-RPE phagocytosed photoreceptor outer segments and displayed transient increases in intracellular calcium levels after ATP stimulation (89.9± 8.2 nM), similar to human prenatal RPE cultures (~100 nM).
After passaging and expansion, hiPSC-RPE continued to display key cell-specific properties that were comparable to cultured human prenatal RPE. Our findings underscore the capacity of hiPSCs to serve as a versatile source of patient-specific RPE for the study and potential treatment of retinal diseases.
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