Purpose: Genome-wide association studies have recently uncovered many loci associated with variation in intraocular pressure (IOP). Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs) and thus, IOP regulation.
Design: Experimental study.
Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent.
View Article and Find Full Text PDFAge-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development.
View Article and Find Full Text PDFTo 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.
View Article and Find Full Text PDFThere 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.
View Article and Find Full Text PDFApolipoprotein 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, β amyloid, and Tau phosphorylation levels.
View Article and Find Full Text PDFBackground: The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) has provided a foundation for in vitro human disease modelling, drug development and population genetics studies. Gene expression plays a critical role in complex disease risk and therapeutic response. However, while the genetic background of reprogrammed cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of individual cells which would provide significant resolution.
View Article and Find Full Text PDFLeber hereditary optic neuropathy (LHON) is one of the most common primary mitochondrial diseases. It is caused by point mutations in mitochondrial DNA (mtDNA) genes and in some cases, it can result in irreversible vision loss, primarily in young men. It is currently unknown why LHON mutations affect only some carriers and whether bioenergetic compensation enables unaffected carriers to overcome mitochondrial impairment and preserve cellular function.
View Article and Find Full Text PDFPurpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.
View Article and Find Full Text PDFWe 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.
View Article and Find Full Text PDFThe CRISPR/Cas system could provide an efficient and reliable means of editing the human genome and has the potential to revolutionize modern medicine; however, rapid developments are raising complex ethical issues. There has been significant scientific debate regarding the acceptability of some applications of CRISPR/Cas, with leaders in the field highlighting the need for the lay public's views to shape expert discussion. As such, we sought to determine the factors that influence public opinion on gene editing.
View Article and Find Full Text PDFBiochim Biophys Acta Mol Cell Biol Lipids
July 2018
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.
View Article and Find Full Text PDFWe report the generation of the hiPSC line CERAi001-A-6 from primary human dermal fibroblasts. Reprogramming was performed using episomal vector delivery of OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA for p53.
View Article and Find Full Text PDFPluripotent stem cells are an extremely powerful tool in modeling human diseases and hold much promise for personalized regenerative or cell replacement therapies. There is an increasing need for reproducible large-scale stem cell and differentiated progeny production, with minimal variation, rendering manual approaches impracticable. Here, we provide an overview of systems currently available for automated stem cell culture, and undertake a review of their capacities, capabilities, and relative limitations.
View Article and Find Full Text PDFCybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.
View Article and Find Full Text PDFEpigenetic variation is implicated in a range of non-communicable diseases, including those of the eye. However, investigating the role of epigenetic variation in central tissues, such as eye or brain, remains problematic and peripheral tissues are often used as surrogates. In this study, matched human blood and eye tissue (n = 8) were obtained post-mortem and DNA methylation profiling performed on blood, neurosensory retina, retinal pigment epithelium (RPE)/choroid and optic nerve tissue using the Illumina Infinium HumanMethylation450 platform.
View Article and Find Full Text PDFPatient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modeling, and drug discovery. However, the processes of reprogramming, maintenance, and differentiation are labor intensive and subject to intertechnician variability. To address these issues, we established and optimized protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO.
View Article and Find Full Text PDFNon-healing chronic wounds present a major biological, psychological, social, and financial burden on both individual patients and the broader health system. Pathologically extensive inflammation plays a major role in the disruption of the normal healing cascade. The causes of chronic wounds (venous, arterial, pressure, and diabetic ulcers) can be examined through a juxtaposition of normal healing and the rogue inflammatory response created by the common components within chronic wounds (ageing, hypoxia, ischaemia-reperfusion injury, and bacterial colonisation).
View Article and Find Full Text PDFOngoing breakthroughs with CRISPR/Cas-based editing could potentially revolutionize modern medicine, but there are many questions to resolve about the ethical implications for its therapeutic application. We conducted a worldwide online survey of over 12,000 people recruited via social media to gauge attitudes toward this technology and discuss our findings here.
View Article and Find Full Text PDFReprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis.
View Article and Find Full Text PDFBackground. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes.
View Article and Find Full Text PDFWe demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells.
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