Ionic Signaling in Retinal Pigment Epithelium: From physiological characterization to stem cell applications

Retinal pigment epithelium (RPE) is crucial for our visual ability performing important functions for the maintenance of photoreceptor viability. Correct physiological functionality is a prerequisite for RPE to execute all its retinal maintenance tasks, several of which are dependent on the various ion channels it expresses. Malfunctions in RPE physiology often cause vision-threatening retinal diseases that can eventually lead to blindness. Stem cell-derived RPE provides a potential model system to investigate RPE physiology and pathophysiology while serving as a cell source for cell transplantation therapies to replace the diseased RPE.

The aim of this dissertation was to investigate the physiology of human embryonic stem cell (hESC)-derived RPE concentrating on ionic signaling and to evaluate these features compared to the native counterpart. This aim was approached by determining the distribution of different ion channels with immunostainings and confocal microscopy in the hESC- and native mouse RPE. The functionality of the ion channels in these cells was studied with patch clamp electrophysiology by recording ionic currents across the cell membrane. The physiological role of ion channels was evaluated for two key processes in the RPE: 1) the phagocytosis of the photoreceptor outer segments (POS) by performing phagocytosis assays and 2) the secretion of the vascular endothelial growth factor (VEGF) using ELISA. These assays were combined with manipulation of ion channel activity with pharmacological compounds. Computational modeling was used to investigate the intra- and intercellular Ca2+ signaling in the RPE monolayer.

In this thesis, a diverse pattern of Ca2+, K+, and Cl- channels were detected at the protein level from the hESC-RPE cell membrane, and generally, the ion channel localization profile was comparable to the native mouse RPE. The basolateral localization of inwardly rectifying K+ channel Kir7.1 and the apical localization of L-type voltage-gated Ca2+ channel CaV1.3 were found here as novel observations in the RPE. Furthermore, the work revealed a link between CaV1.3 and RPE development as the CaV1.3 channel localization changed remarkably during RPE maturation. Considering physiological relevance, the L-type Ca2+ channels participated in the regulation of phagocytosis and VEGF secretion. The features of L-type Ca2+ currents recorded from the hESC-RPE resembled those measured from the native mouse RPE in this study as well as the native RPE reported in the literature. However, differences appeared between K+ currents in the hESC-RPE compared to the literature from the native RPE, especially with the Kir currents. This may indicate a compromised functionality of Kir channels in the hESC-RPE.

In conclusion, ion channels are highly sensitive to the external cell culture conditions as well as the maturation level of the RPE. Thus, assessing the functional phenotype of the stem cell-derived RPE is extremely important when evaluating the physiological maturity of the cells and their capability to perform the crucial tasks of the native RPE. All in all, the presence of functional machinery of ion channels in the hESC-RPE indicates great potential for their use as cell models and in transplantation therapies.