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

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.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 p27(Kip1) 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.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

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study

Employing Nanosafety Standards in a Nanomaterial Research Environment: Lessons Learned and Refinement Potential

Extensive research is currently being conducted on nanotechnologies worldwide, and the applications of nanomaterials are continuously expanding. Given their unique intrinsic characteristics, such as their small size and increased reactivity, nanomaterials may pose an occupational, environmental or consumer hazard. Therefore, a highly important aspect of ensuring the sustainable use of nanotechnologies is the establishment of proper health and safety practices. The area of nanosafety research has produced significant outcomes the last decades, and many of these achievements have been reflected in the standardization field. In this work, a discussion of prominent nanosafety standards (ISO/TS 12901-2:2014 and ISO/TR 12885:2018) is presented, based on the barriers faced during the endeavor to apply their principles within a research context. A critical viewpoint regarding their application is presented, and gaps faced in adapting the standards to the materials and processes applied are noted. Additionally, approaches that were followed to circumvent these gaps are also highlighted as suggestions to potentially overcome these barriers in future standardization efforts

Employing Nanosafety Standards in a Nanomaterial Research Environment: Lessons Learned and Refinement Potential

Extensive research is currently being conducted on nanotechnologies worldwide, and the applications of nanomaterials are continuously expanding. Given their unique intrinsic characteristics, such as their small size and increased reactivity, nanomaterials may pose an occupational, environmental or consumer hazard. Therefore, a highly important aspect of ensuring the sustainable use of nanotechnologies is the establishment of proper health and safety practices. The area of nanosafety research has produced significant outcomes the last decades, and many of these achievements have been reflected in the standardization field. In this work, a discussion of prominent nanosafety standards (ISO/TS 12901-2:2014 and ISO/TR 12885:2018) is presented, based on the barriers faced during the endeavor to apply their principles within a research context. A critical viewpoint regarding their application is presented, and gaps faced in adapting the standards to the materials and processes applied are noted. Additionally, approaches that were followed to circumvent these gaps are also highlighted as suggestions to potentially overcome these barriers in future standardization efforts

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

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

Quantitative Real-Time PCR analysis of gene expression in peripheral and central RPE tissue and cell cultures.

<p>Gene expression of p27^kip1 (Ai.), Cyclin D1 (Bi.), and mTOR (Ci.), in RPE tissue obtained from the periphery and centre of adult mouse RPE. Gene expression of p27^kip1 (Aii.), Cyclin D1 (Bii.), and mTOR (Cii.) in cultured cells obtained from the periphery and centre of adult mouse RPE. Graphs show relative gene expression levels from independent samples normalized to ACTB (at least 6 independent samples obtained from each region); Mann-Whitney Test, * P<0.05 in each case.</p