Efflux Protein Expression in Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy

Using human induced pluripotent stem cells to treat retinal disease

AbstractThe eye is an ideal target for exploiting the potential of human induced pluripotent stem cell (hiPSC) technology in order to understand disease pathways and explore novel therapeutic strategies for inherited retinal disease. The aim of this article is to map the pathway from state-of-the art laboratory-based discoveries to realising the translational potential of this emerging technique. We describe the relevance and routes to establishing hiPSCs in selected models of human retinal disease. Additionally, we define pathways for applying hiPSC technology in treating currently incurable, progressive and blinding retinal disease

Effects of lipids and lipid derivatives on undifferentiated growth of human embryonic stem cells

Verkkojulkaisu salainen 1.6.2015 saakk

Effects of lipids and lipid derivatives on undifferentiated growth of human embryonic stem cells

Verkkojulkaisu salainen 1.6.2015 saakk

Localization of ATP-dependent efflux transporter proteins. A-L

<p>) Confocal micrographs after indirect immunofluorescence labeling with efflux pump proteins MRP-1, -4, or -5 (green), and eye-specific proteins MITF and cellular retinaldehyde-binding protein (CRALBP, both red), the polarization marker Na⁺/K⁺ ATPase (red), and the nuclear label 4′,6′-diamidino-2-phenylidole (blue). In figures <b>M-P</b>) the brightfield micrographs show the same ARPE-19 cells and fusiform, early cobblestone, and cobblestone hESC-RPE as shown in the confocal images. Scale bars, 10 µm.</p

Functional testing of ATP-dependent efflux transporter proteins and viability of cultured cells. A

<p>) Calcein retention in ARPE-19, undifferentiated hESC, fusiform, and cobblestone hESC-RPE, and hFF cells in the presence or absence ( =  control) of efflux protein inhibitors. Retention is expressed as a percentage of fluorescence relative to control (control = 100%). The studies were repeated at least three times for ARPE-19 and fusiform hESC-RPE, and once each for undifferentiated hESC, cobblestone hESC-RPE, and hFF. Data are expressed as mean±SD, *p<0.05, **p<0.01, ***p<0.001. B-E) Representative images of viable (green fluorescence) and dead (red fluorescence) ARPE19 (B,C) and fusiform hESC RPE cells (D,E). The scale bar 100 µM.</p

Expression of ATP-dependent efflux transporter genes.

<p>Relative expression of MRP1, MRP3, MRP4, MRP5, P-gp, and MRP6 genes. D407 used as reference sample for all genes except MRP-6, for which HEK-293 was used instead. Values that are significantly different from those of the reference sample are marked with an asterisk (*). For better visualization, fold-change is represented on a logarithmic scale. Standard deviations of fold-change from three separate experiments are presented as error bars.</p

Morphology and gene expression of hESC on different maturation stages.

<p>Brightfield micrographs of cell cultures showing the representative morphology of <b>A</b>) undifferentiated hESC (Regea08/017), <b>B</b>) fusiform hESC-RPE, <b>C</b>) epithelioid hESC-RPE, <b>D</b>) cobblestone hESC-RPE. Scale bars, 100 µm, <b>E</b>) Gene expression of <b>1</b>: D407, <b>3</b>: ARPE-19, <b>5</b>: undifferentiated hESC, <b>7</b>: fusiform hESC-RPE, <b>9</b>: epithelioid hESC-RPE, <b>11</b>: cobblestone hESC-RPE, <b>13</b>: hFF. –RT- negative controls (i.e., samples not treated with reverse transcriptase) are placed adjacent to each sample in the same order: <b>2</b>: D407, <b>4</b>: ARPE-19, <b>6</b>: undifferentiated hESC, <b>8</b>: fusiform hESC-RPE, <b>10</b>: epithelioid hESC-RPE, <b>12</b>: cobblestone hESC-RPE, <b>14</b>: hFF. <b>F</b>) Culture periods of the studied samples in all analyses. Cells were selected based on their morphology rather than the culture period.</p