Mature Peripheral RPE Cells Have an Intrinsic Capacity to Proliferate; A Potential Regulatory Mechanism for Age-Related Cell Loss

A Potential Regulatory Mechanism for Age-Related Cell Loss. PLoS ONE 6(4): e18921. doi:10.1371/journal.pone.0018921 Mature Peripheral RPE Cells Have an Intrinsic Capacity to Proliferate; A Potential Regulatory Mechanism for Age-Related Cell Loss Ioannis Kokkinopoulos 0 Golnaz Shahabi 0 Alan Colman 0 Glen Jeffery 0 Mike O. Karl, Center for Regenerative Therapies Dresden, Germany 0 1 Institute of Ophthalmology, University College London , London , United Kingdom , 2 School of Biomedical and Health Sciences Wolfson Centre for Age-Related Diseases, King's College London , London , United Kingdom , 3 Singapore Stem Cell Consortium, Singapore, Singapore Background: Mammalian peripheral retinal pigmented epithelium (RPE) cells proliferate throughout life, while central cells are senescent. It is thought that some peripheral cells migrate centrally to correct age-related central RPE loss. Methodology/Principal Findings: We ask whether this proliferative capacity is intrinsic to such cells and whether cells located centrally produce diffusible signals imposing senescence upon the former once migrated. We also ask whether there are regional differences in expression patterns of key genes involved in these features between the centre and the periphery in vivo and in vitro. Low density RPE cultures obtained from adult mice revealed significantly greater levels of proliferation when derived from peripheral compared to central tissue, but this significance declined with increasing culture density. Further, exposure to centrally conditioned media had no influence on proliferation in peripheral RPE cell cultures at the concentrations examined. Central cells expressed significantly higher levels of E-Cadherin revealing a tighter cell adhesion than in the peripheral regions. Fluorescence-labelled staining for E-Cadherin, F-actin and ZO-1 in vivo revealed different patterns with significantly increased expression on central RPE cells than those in the periphery or differences in junctional morphology. A range of other genes were investigated both in vivo and in vitro associated with RPE proliferation in order to identify gene expression differences between the centre and the periphery. Specifically, the cell cycle inhibitor p27Kip1 was significantly elevated in central senescent regions in vivo and mTOR, associated with RPE cell senescence, was significantly elevated in the centre in comparison to the periphery. Conclusions: These data show that the proliferative capacity of peripheral RPE cells is intrinsic and cell-autonomous in adult mice. These differences between centre and periphery are reflected in distinct patterns in junctional markers. The regional proliferation differences may be inversely dependent to cell-cell contact. - Funding: This work was supported by an MRC grant [the Medical Research Council UK (G03000341)] and The Rosetree Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The retinal pigmented epithelium (RPE) is a monolayer wrapping around the outer retina and forms part of the blood retinal barrier. It is critical for normal development of the adjacent neural retina [1] and at maturity sustains outer retinal function [2,3,4]. Both the RPE and the neural retina develop with a centre to periphery gradient, such that the last cells to leave the cell cycle are in the periphery [5,6]. The far periphery is also thought to be a region from which stem cells can be harvested in the adult [7]. Mature peripheral RPE cells retain an ability to divide throughout life and some migrate into central senescent regions [8,9]. Here we ask whether these peripheral cells have an intrinsic capacity to divide, marking them as distinct, or whether this ability is related to their local microenvironment and/or its signals. We examine RPE proliferative abilities in vitro from cells harvested from central and peripheral retinal regions and determine if central cells are able to impose senescence upon peripheral cells via soluble signals by exposing them to medium conditioned centrally. We also ask whether there is differential expression between the centre and periphery of key targeted genes involved in cell cycle activity and cell migration that may underpin their different abilities. In some non-mammalian vertebrates, RPE proliferation can be a step towards trans-differentiation and the building of a new retina, although such abilities have not been explored in mammals [10]. The long term aim of this study is to identify the key factors that distinguish those RPE cells that are able to trans-differentiate from those that are not. Peripheral RPE cells proliferate in vivo Mouse RPE was examined following in vivo BrdU injections to confirm that RPE cells proliferate in this species. BrdU positive cells were present, but only in the periphery, consistent with previous studies [9] (Figure 1A, 1B). The only difference between Figure 1. Peripheral RPE cells proliferate in vivo and in vitro. 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 mm. N (number of eyes examined) = 3. doi:10.1371/journal.pone.0018921.g001 the current results and previous studies was that fewer cells were identified in mouse than rat. Peripheral RPE cells proliferate more than central RPE cells in vitro The vast majority of cultured cells, irrespective of origin, were positive for Otx2 [11], confirming their identity as RPE cells (Figure 1C), with some co-expressing BrdU. All BrdU+ cells were also Otx2+. Low density RPE cultures were established from central and peripheral retinal regions and from the complete RPE sheet and exposed to BrdU. Significantly more proliferating cells were found in cultures derived from the periphery than the centre (Figure 2A, 2B, 2C). When whole RPE sheets were used and levels of proliferation assessed, they were different from those in the periphery but not from those from the centre (Figure 2C). Cells in these experiments had been cultured for 9 days and it is possible that differences in proliferation between central and peripheral areas were much smaller originally, but that over this period the pool of proliferating ce (...truncated)