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

Reverse-transcriptase–PCR primer sequences and used annealing temperatures.

<p>Reverse-transcriptase–PCR primer sequences and used annealing temperatures.</p

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

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

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