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Age-related macular degeneration (AMD), the most common cause of vision loss with ageing, is characterised by degeneration of the photoreceptors and retinal pigment epithelium (RPE) and changes in the extracellular matrix (ECM) underlying the RPE. The pathogenesis of AMD is still not fully understood. In this study we investigated the in vitro growth and function of primary human RPE cells in response to different ECM substrates, including nitrite-modified ECM. We initially confirmed the presence of disorganised retinal glial and photoreceptor cells, marked retinal cytoplasmic and Bruch's membrane expression of nitro-tyrosine (an oxidative stress marker) and increased numbers of Iba1+ macrophages/microglia in human donor eye sections (aged and AMD) using multi-marker immunohistochemistry (n = 3). Concurrently, we utilised two-photon microscopy to reveal topographical changes in flatmounts of RPE-associated ECM and in the underlying choroid of aged and AMD donor eyes (n = 3). To recapitulate these observations in vitro, we then used primary human RPE cells to investigate how different ECM proteins, including nitrite cross-linked RPE-secreted ECM, modified RPE cell growth and function. Collagen I or IV increased RPE attachment and spreading two-to three-fold, associated with significantly increased cell migration and proliferation, consistent with a preferential interaction with these matrix substrates. Primary human RPE cells grown on collagen I and IV also showed increased secretion of pro-inflammatory cytokines, MCP-1 and IL-8. Nitrite-modification of RPE-secreted ECM (simulating ageing of Bruch's membrane) significantly reduced in vitro RPE attachment to the ECM and this was mitigated with collagen IV coating of the modified ECM. Taken together, our observations confirm the importance of RPE-ECM interactions for normal RPE growth and function, and for inducing RPE secretion of pro-inflammatory cytokines. Furthermore, the findings are consistent with ageing and/or oxidative stress-induced disruption of RPE-ECM interactions contributing to the pathogenesis of AMD.

The choroid within the human eye contains a rich milieu of cells including melanocytes. Human choroidal melanocytes (HCMs) absorb light, regulate free radical production, and were recently shown to modulate inflammation. This study aimed to identify key genes and pathways involved in the inflammatory response of HCMs through the use of RNA-seq. Primary HCMs were cultured from donor choroids, RNA was extracted from control and lipopolysaccharide (LPS)-treated HCMs, and mRNA was sequenced. Functional annotation and pathway analysis were performed using gene ontology and gene set enrichment analyses. Representative RNA-seq results were verified with RT-qPCR and protein measurements. We detected 100 differentially expressed genes including an array of CCL and CXCL cytokines and mediators of cell-cell and cell-matrix adhesion, such as ICAM1, CLDN1, CCN3, ITGA1 and ITGA11. Functional annotation showed that these gene sets control inflammatory pathways, immune cell trafficking, cell-cell adhesion, interactions with the extracellular matrix and blood vessels, angiogenesis and epithelial-to-mesenchymal transitions. Our study provides insights into the transcriptional regulation of primary HCMs in response to inflammatory stimuli and identifies novel melanocyte-driven mechanisms potentially involved in choroidal homeostasis and inflammation.

Toll-like receptors (TLRs) are a class of pattern recognition receptors that sense highly conserved pathogen associated antigenic determinants, triggering an innate immune response and subsequently instructing the adaptive immune system so that together, the pathogen can be eliminated. TLRs are widely distributed in human ocular tissues and cell types, and are active players in ocular inflammation. To date, the presence and function of TLRs on human choroidal melanocytes (HCMs), the most abundant choroidal cell type, have not been characterized. The current study investigated the in vitro and in situ expression and functional status of TLRs on HCMs. HCMs were isolated and cultured from post-mortem human donor eyes, and displayed characteristic melanocyte morphology and MART1 expression – a key melanocyte lineage marker up to passage 5 (P5). In vitro experiments used P1 to P4 HCMs from different donor eyes. Initial quantitative real-time PCR (qPCR) analysis revealed that HCMs (n = 3 donors) expressed specific mRNA transcripts for TLR1-10 and MYD88 (a key adaptor protein initiating the TLR signalling pathway). HCMs were stimulated with a set of synthetic TLR specific agonists and the secretion of pro-inflammatory cytokines, MCP-1 and IL-8, at 24 h measured by ELISA (n = 3 donors). The agonists Pam3CSK4 (TLR1/2), Poly I:C (TLR3), LPS (TLR4), Flagellin (TLR5), and FLS-1 (TLR2) induced a significant increase in the production of MCP-1 and IL-8, compared to untreated cells. Application of biotinylated Pam3CSK4 provided in vitro visualization of receptor-agonist interactions for TLR1/2. We confirmed that cultured HCMs (n = 3 donors) expressed TLR1-6 protein using immunocytochemistry and confocal microscopy. The expression and distribution of TLR 1–6 was also studied in human choroid and retinal pigment epithelium (RPE) sections (n = 3 eyes) using immunofluorescence and confocal microscopy. Strong TLR1-6 immunolabelling that co-localized with melanocyte-dense areas (and RPE) was consistently observed; intraluminal and blood vessel-related cells (including endothelial cells) also expressed several TLRs. Taken together these observations show for the first time that HCMs constitutively express a range of functional TLRs, and as such can contribute to choroidal responses during infection and inflammation.

During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68+ microglia and astrocytes mediate phagocytosis during target-dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple-marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live-cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum-free environment, doublecortin+ (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7highDCXlow hNPCs and P2X7highDCXhigh neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum-free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target-dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor. Stem Cells 2015;33:526–541

Stem cells prelabelled with iron oxide nanoparticles can be visualised using magnetic resonance imaging (MRI). This technique allows for noninvasive long-term monitoring of migration, integration and stem cell fate following transplantation into living animals. In order to determine biocompatibility, the present study investigated the biological impact of introducing ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) into primary human fetal neural precursor cells (hNPCs) in vitro. USPIOs with a mean diameter of 10-15 nm maghemite iron oxide core were sterically stabilised by 95% methoxy-poly(ethylene glycol) (MPEG) and either 5% cationic (NH2) end-functionalised, or 5% Rhodamine B end-functionalised, polyacrylamide. The stabilising polymer diblocks were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation. Upon loading, cellular viability, total iron capacity, differentiation, average distance of migration and changes in intracellular calcium ion concentration were measured to determine optimal loading conditions. Taken together we demonstrate that prelabelling of hNPCs with USPIOs has no significant detrimental effect on cell biology and that USPIOs, when utilised at an optimised dosage, are an effective means of noninvasively tracking prelabelled hNPCs.

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.