Aquaporin Expression and Function in Human Pluripotent Stem Cell–Derived Retinal Pigmented Epithelial Cells

Abstract

PURPOSE. Aquaporins (AQPs), a family of transmembrane water channel proteins, are essential for allowing passive water transport through retinal pigmented epithelial (RPE) cells. Even though human native RPE cells and immortalized human RPEs have been shown to express AQPs, the expression of AQPs during the differentiation in stem cell-derived RPE remains to be elucidated. METHODS.In human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs)-derived RPE cells, the expression of several AQPs was determined by quantitative real-time PCR and the localization of AQP1 was assessed with confocal microscopy. The functionality of AQP water channels was determined by cell volume assay in hESC-derived RPE cells.RESULTS. AQP1, AQP3, AQP4, AQP5, AQP6, AQP7, AQP10, AQP11, and AQP12 were expressed in hESC-and hiPSC-derived RPE cells. Furthermore, the expression of AQP1 and AQP11 genes were significantly upregulated during the maturation of both hESC and iPSC into RPE. Confocal microscopy shows the expression of AQP1 at the apical plasma membrane of polarized cobblestone hESC-and hiPSC-derived RPE cells. Lastly, aquaporin inhibitors significantly reduced AQP functionality in hESC-RPE cells.CONCLUSIONS. hESC-RPE and hiPSC-RPE cells express several AQP genes, which are functional in mature hESC-derived RPE cells. The localization of AQP1 on the apical plasma membrane in mature RPE cells derived from both hESC and hiPSC suggests its functionality. These data propose that hESC-and hiPSC-derived RPE cells, grown and differentiated under serum-free conditions, resemble their native counterpart in the human eye.

Autophagy Regulates Proteasome Inhibitor-Induced Pigmentation in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells

“…Nonetheless, hESC-RPE cells differ from ARPE-19 cells in their gene expression [35] and pigmentation [56], as well as aggregation levels under proteasome inhibition [2,3,5]. In this study, we show that proteasome inhibition leads to high accumulation of melanosomes in hESC-RPE cells, a finding in line with previous documentations [28,52,53,57]. The lower calcein fluorescence intensity detected in live/dead viability assay is in line with the accumulation of melanosomes in MG-132- and MG-132 + bafilomycin A1-treated samples.…”

Comparative proteomic analysis of human embryonic stem cell-derived and primary human retinal pigment epithelium

“…Important transporters and ion channels were identified in the hESC-RPE such as Na + K + -transporting ATPase subunits alpha-1, and beta-1 and −2 (ATP1A1, ATP1B1) localized at the apical surface of the RPE as shown previously for our hESC-RPE 18 . Aquaporin 1 (fragment) (AQP1), channel protein that mediates water transport, is apically expressed both in human foetal RPE primary cultures and in hESC-RPE cells 19 . Proteomic analyses of the plasma membrane after subcellular fractionation could provide further insides into expression of transporter and channel proteins by the hPSCs.…”

Plasma membrane protein polarity and trafficking in RPE cells: Past, present and future

“…intestine and lung, where CFTR is apically localized (Levin and Verkman, 2006). Water transport is mediated by Aquaporin-1 water channels, which have been found to be apically expressed both in hfRPE primary cultures (Stamer et al, 2003), and more recently in stem cell-derived RPE cells (Juuti-Uusitalo et al, 2013). The localization of the Na,K-ATPase in RPE cells is also unconventional, as it is localized to the apical surface, rather than to the basolateral PM, the characteristic localization of the sodium pump in most epithelia (Bok, 1982; Gundersen et al, 1991; Ostwald and Steinberg, 1980; Rizzolo, 1990).…”