Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.

The pandemic's impact on a lymphoedema service: reflections of a lymphoedema clinical lead nurse specialist.

Louise Rooney has worked in the NHS for 34 years in various roles, but her passion is the treatment of lymphoedema and how best to support patients with this underestimated and debilitating chronic condition. She has been working as a lymphoedema clinical nurse specialist since 2009; in 2019, she became the lymphoedema clinical lead at the Walsall Palliative Care Centre, Walsall Healthcare NHS Trust. In this article, Rooney elaborates on the implications of the COVID-19 pandemic on the lymphoedema service, her own practice and that of her colleagues.

Culture Variabilities of Human iPSC-Derived Cerebral Organoids Are a Major Issue for the Modelling of Phenotypes Observed in Alzheimer's Disease.

Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer's disease (AD), with the presence of APOE-ε4 associated with increased risk of developing AD. Here, we reprogrammed human fibroblasts from individuals with different APOE-ε genotypes into induced pluripotent stem cells (iPSCs), and generated isogenic lines with different APOE profiles. Following characterisation of the newly established iPSC lines, we used an unguided/unpatterning differentiation method to generate six-month-old cerebral organoids from all iPSC lines to assess the suitability of this in vitro system to measure APOE, ß amyloid, and Tau phosphorylation levels. We identified variabilities in the organoids' cell composition between cell lines, and between batches of differentiation for each cell line. We observed more homogenous cerebral organoids, and similar levels of APOE, ß amyloid, and Tau when using the CRISPR-edited APOE isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-ε4/ε4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-ε status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the cell-line-to-cell-line and batch-to-batch variabilities need to be considered when using cerebral organoids.

Retinal ganglion cell-specific genetic regulation in primary open-angle glaucoma.

To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared with those from healthy individuals. We performed single-cell RNA sequencing of a total of 247,520 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 4,443 significant loci across all cell types, 312 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 97 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.

Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media.

We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlex and TeSR-E8 media. Analysis of transcriptome of single cells revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Analysis of individual cells from four samples consisting of two different iPSC lines each grown in the two culture media revealed a shared subpopulation structure with three main subpopulations different in pluripotency states. By implementing a machine learning approach, we estimated that most cells within each subpopulation are very similar between all four samples. The single-cell RNA sequencing analysis of iPSC lines grown in both media reports the molecular signature in StemFlex medium and how it compares to that observed in the TeSR-E8 medium.

Compression therapy and exercise: enhancing outcomes.

Compression therapy is the main method used within the treatment and management of lymphoedema and chronic oedema. The increasing prevalence of the condition, which has multiple causes, such as genetic factors, age and external factors, require the effective management of the condition, and to enhance the management methods used to contain the condition. The use of exercise alongside the mainstay method of treatment (compression therapy) has been an underutilised area. The application of a structured exercise programme in conjunction with multi-layer lymphoedema bandaging combined with an adjustable velcro wrap based systems led to a decrease in limb volume in one case study. The results of the programme indicate that the approach is beneficial and requires embedding further within Walsall lymphoedema service, due to improved patient outcomes, and cost effectiveness in terms of resources.

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors.

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.