Résumé
Purpose : Induced pluripotent stem cells (iPSC) have revolutionized the study of human diseases as they can renew indefinitely, undergo multi-lineage differentiation and serve to generate disease-specific models. In particular, iPSC-derived retinal pigment epithelium (RPE) has been shown to be a powerful tool for pathophysiological and therapeutic studies, as it is morphologically and functionally characteristic of the RPE in vivo. The functions reproduced in vitroby this tissue include phagocytosis, fluid transport and growth factor secretion, which are regulated by variations in intracellular calcium levels and require the presence of functional ion channels. In our current study, we demonstrate the presence of functional calcium, potassium and chloride channels in human iPSC-derived RPE.Methods : Wild type iPSC-derived RPE monolayer was grown to passage 3 and the expression of the genes coding for L-type calcium channels (CaV1.1, CaV1.2 and CaV1.3), T-type calcium channels (CaV3.1 and CaV3.3), potassium channels (MaxiK, Kir 4.1 and Kir7.1) and the chloride channel CLC2 were tested by RT-PCR. Immunofluorescence studies using antibodies specific to the same channels were used to verify protein expression. FURA-2 live imaging following stimulation by ATP was used to test variations in intracellular calcium levels. Patch clamp studies were used to determine the functionality of the three types of channels.Results : The expression of all the channels was detected by RT-PCR. Immunofluorescence studies confirmed the expression of each channel and determined their specific subcellular localization: CaV1.1 and CaV1.3 basolateral; Cav3.1 and CaV3.3 apicolateral and/or microvilli; Maxi K basolateral; Kir4.1 apical; Kir 7.1 microvilli, and CLC2 apicolateral. Both CaV1.3 and CLC2 also co-localised with the primary RPE cilium. Electrophysiological responses for all three classes of ion channels were recorded by patch-clamp analysis on whole cells. We are currently studying the effect of specific channel inhibition on RPE-specific functions.Conclusions : To our knowledge, we demonstrate for the first time the presence of functional calcium, potassium and chloride channels on iPSC-derived RPE. This further validates this model as a bona fidetissue for physiological and pathophysiological studies. Moreover, these results have important implications in an era where iPSC-derived RPE transplantation is already in the clinic.