Peripheral, central and total RPE cell cultures have different proliferation profiles.

<p>Peripheral, central and total RPE cells were cultured for 9 days then received a single 4-hour BrdU pulse prior to fixation. Representative microphotographs of BrdU-labelled cultures of (A) peripheral RPE cells and (B) central RPE cells. White arrows indicate pigmented BrdU⁺ cells. (C) A graph indicating the number of BrdU⁺ cells in cultures harvested from each region (Peripheral: circles. Central: squares. Total: triangles). Mann-Whitney Test; *p = 0.02, **p = 0.04. No statistical significance was found between central and total RPE BrdU⁺ cells counts. Error bars = SEM. Peripheral and central RPE cells were cultured as above for 3 days, fixed and immuno-stained with Ki67. Representative microphotographs of Ki67⁺ cells of (D) peripheral cells and (E) central cells. White arrows indicate Ki67⁺ cells. The number of Ki67⁺ cells counted from each region is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018921#pone-0018921-g002" target="_blank">Figure 2F</a> where cell densities in the two cultures were similar (Periphery: circle points. Central; square points). Mann-Whitney Test; *p = 0.05. Error bars = SD. In A–C, when labelled cell numbers are normalised against the total cell number for the two regions the differences remain significantly different (Mann-Whitney Test; BrdU⁺/total cell number - Periphery against Centre; p = 0.037 and Ki67⁺/total cell number - Periphery against Centre; p = 0.0237). In vivo protein expression of active (phosphorylated) beta-catenin in peripheral and central RPE (G). N = 3 eyes. Scale bars = 5 µm.</p

Peripheral RPE cells proliferate in vivo and in vitro.

<p>The adult mouse RPE was investigated to confirm it contains a peripheral region where cell division occurs. Animals were given daily BrdU injections for 5 consecutive days and were sacrificed 1, 2, 3 and 4 weeks after the last injection day. (A) Photomicrograph of the RPE flatmount indicating, in the circle, the peripheral region in which BrdU⁺ RPE cells were found. The dotted white line illustrates the peripheral and central RPE zones. (B) Higher magnification showing cells positive for BrdU (in red). (C) RPE cells were cultured for 9 days and on final day they received a single 4-hour BrdU pulse prior to fixation. The majority of cells expressed Otx2 (RPE-specific marker, red), with a number of cells also co-expressing BrdU (green). Scale bar = 5 µm. N (number of eyes examined) = 3.</p

Schematic graph of the adult mouse RPE and characteristics of peripheral and central RPE cells.

<p>Peripheral and Central RPE cells express different levels of genes that play crucial role in cell proliferation. The levels of protein and gene expression for key cell adhesion molecules is also different between these two RPE populations, indicating distinct cell-cell contact behaviour.</p

Central RPE cells do not inhibit peripheral RPE cells from proliferating via diffusible signals.

<p>RPE cultures were setup from peripheral and central RPE and were allowed to proliferate at low density for one week, before peripheral RPE cultures were introduced to central RPE medium for two days before a 4-hour BrdU pulse. (A) The graph indicates the number of BrdU⁺ cells per region per total cell number. Periphery (circle points), central (square points) and control periphery (triangle points). Cell number per culture; 5,000 cells/well. Mann-Whitney Test; *p = 0.03. **p = 0.02. Peripheral RPE versus control RPE periphery was not found to be statistically significant (p = 0.68). Error bars = SD. (B) RPE cultures were setup from peripheral and central RPE and allowed to proliferate at high density for one week, before peripheral RPE cultures were introduced to central RPE medium for two days before a 4-hour BrdU pulse. The graph indicates the number of BrdU⁺ cells per region. Periphery (circle points), central (square points) and control periphery (triangle points). Cell number per culture; 10,000 cells/well. Error bars = SD.</p