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Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that they retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 causes a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.

ImportancePrevious studies identify putative genes associated with diabetic retinopathy only focusing on specific clinical stage, thus resulting genes are not necessarily reflective of disease progression.ObjectiveTo identify genes associated with the severity level of diabetic retinopathy using likelihood-ration test (LRT) and ordinal logistic regression (OLR) model, as well as to profile immune and retinal cell landscape in progressive diabetic retinopathy using a machine learning deconvolution approach.Design, Setting, and ParticipantsThis study used published transcriptomic dataset (GSE160306) from macular regions of donors with different degrees of diabetic retinopathy (10 healthy controls, 10 cases of diabetes, 9 cases of nonproliferative diabetic retinopathy and 10 cases of proliferative diabetic retinopathy or combined with diabetic macular edema). Transcriptomic dataset was released on April 19, 2021; data were analyzed on 28th March 2022.Main Outcomes and MeasuresIdentification of severity-associated genes with LRT and OLR model and proportional changes of immune and retinal cells in progressive diabetic retinopathy.ResultsBy controlling for gender and age using LRT and OLR, 50 genes were identified to be significantly increased in expression with the severity of diabetic retinopathy. Functional enrichment analyses suggested these severity-associated genes are related to inflammation and immune responses. CCND1 and FCGR2B are further identified as key regulators to interact with many other severity-associated genes and are crucial to inflammation. Deconvolution analyses demonstrated that the proportions of memory B cells, M2 macrophages and Müller glia were significantly increased with the progression of diabetic retinopathy.Conclusions and RelevanceThese findings demonstrate the deep analyses of transcriptomic data can advance our understanding of progressive ocular diseases, such as DR, by applying LRT and OLR model as well as bulk gene expression deconvolution.Key PointsQuestionDo genes change in expression or immune cells and retinal cells change in proportions in the human retina with the severity of diabetic retinopathy?FindingsIn this in-depth retinal transcriptomic analysis of 39 participants, consisting of 10 heathy controls, 29 cases of diabetic retinopathy at different clinical stages, severity-associated genes were identified, and the estimated proportions of immune cell and retinal cells were profiled, respectively.MeaningThese findings demonstrate that a group of genes are significantly increased in expression, and the proportions of memory B cells, M2 macrophages and Müller glia were significantly increased with the severity of diabetic retinopathy.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.

Age is the main risk factor for age-related macular degeneration (AMD), a leading cause of blindness in the elderly, with limited therapeutic options. Here we systematically analyzed the transcriptomic characteristics and cellular landscape of the aging retina from controls and patients with AMD. We identify the aging genes in the retina that are associated with innate immune response and inflammation. Deconvolution analysis reveals that the estimated proportion of M2 and M0 macrophages is increased and decreased, respectively with both age and AMD severity. Moreover, we find that Müller glia are increased with age but not with disease severity. Several genes associated with both age and disease severity in AMD, particularly C1s and MR1, are strong positively correlated with the proportions of Müller glia. Our studies expand the genetic and cellular landscape of AMD and provide avenues for further studies on the relationship between age and AMD.

The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.

The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells., A comprehensive analysis of the ocular networks among various tissues is necessary to understand eye physiology in health and disease. Here the authors present a multi-species single-cell transcriptomic atlas consisting of cells of the cornea, iris, ciliary body, neural retina, retinal pigmented epithelium, and choroid.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

Cell therapy offers great promises in replacing the neurons lost due to neurodegenerative diseases or injuries. However, a key challenge is the cellular source for transplantation which is often limited by donor availability. Direct reprogramming provides an exciting avenue to generate specialized neuron subtypes in vitro, which have the potential to be used for autologous transplantation, as well as generation of patient-specific disease models in the lab for drug discovery and testing gene therapy. Here we present a detailed review on transcription factors that promote direct reprogramming of specific neuronal subtypes with particular focus on glutamatergic, GABAergic, dopaminergic, sensory and retinal neurons. We will discuss the developmental role of master transcriptional regulators and specification factors for neuronal subtypes, and summarize their use in promoting direct reprogramming into different neuronal subtypes. Furthermore, we will discuss up-and-coming technologies that advance the cell reprogramming field, including the use of computational prediction of reprogramming factors, opportunity of cellular reprogramming using small chemicals and microRNA, as well as the exciting potential for applying direct reprogramming in vivo as a novel approach to promote neuro-regeneration within the body. Finally, we will highlight the clinical potential of direct reprogramming and discuss the hurdles that need to be overcome for clinical translation.

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines. Keywords: CRISPR, cloning free, gene activation, CRISPR activation, SpCas9, SaCas9, multiplex gene activation, transcriptional activator

In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 ‘A’ strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine (NCT04591717; https://clinicaltrials.gov/ct2/show/NCT04591717), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-β-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-β1 and other pro-inflammatory cytokines. TAK1 is also a key mediator of inflammation, innate immune responses, apoptosis and tissue homeostasis and plays an important role in physiological angiogenesis. Its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. We investigated the regulatory role of TAK1 in pathological angiogenesis in the retina. Transcriptome analysis of human retina featuring retinal neovascularization revealed enrichment of known TAK1-mediated signaling pathways. Selective inhibition of TAK1 activation by 5Z-7-oxozeaenol attenuated aberrant retinal angiogenesis in rats following oxygen-induced retinopathy. Transcriptome profiling revealed that TAK1 activation in human microvascular endothelial cells under TNFα stimulation led to increase the gene expression related to cytokines and leukocyte-endothelial interaction, mainly through nuclear factor kappa B (NFκB) signaling pathways. These results reveal that inhibition of TAK1 signaling may have therapeutic value for the treatment of pathological angiogenesis in the retina.

Aims To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction. Methods and results Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs. Conclusions Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

Conventional methods of neuronal differentiation for human induced pluripotent stem cells (iPSCs) are tedious and complicated, involving multi-stage protocols with complex cocktails of growth factors and small molecules. Artificial extracellular matrix with defined surface topography and chemistry represents a promising venue to improve the neuronal differentiation in vitro. In the present study, we test the impact of a type of colloidal self-assembled patterns called binary colloidal crystals (BCCs) in neuronal differentiation. We developed a CRISPR activation (CRISPRa) iPSC platform that constitutively expresses the dCas9-VPR system, which allows robust activation of the proneural transcription factor NEUROD1 to rapidly induce neuronal differentiation within seven days. We showed that the combinatorial use of BCCs can further improve this neuronal differentiation system. In particular, our results indicate that fine tuning of silica and polystyrene size is critical to generate specific topographies to improve neuronal differentiation and branching. BCCs with 5 μm silica and 100 nm carboxylated polystyrene has the most prominent effect on increasing neurite outgrowth and more complex ramification, while BCCs with 2μm silica and 65nm carboxylated polystyrene is better in promoting neuronal enrichment. These results indicate that biophysical cues can support rapid differentiation and improve neuronal maturation. In summary, our combinatorial approach of CRISPRa and BCCs provides a robust and rapid pipeline for in vitro production of human neurons. Specific BCCs can be adapted to late stages of neuronal differentiation protocols to improve neuronal maturation, which have important implications in tissue engineering, in vitro biological studies and disease modeling.

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >65 genetic risk factors for AMD, including the TMEM97 locus. TMEM97 encodes the Sigma-2 receptor which is involved in apoptosis and cytotoxicity across a range of neurodegenerative diseases. However, the expression pattern of TMEM97 in the human retina and its functional role in retinal cells has remained elusive. Here we utilised CRISPR interference (CRISPRi) to investigate the functional role of TMEM97 in the retina. Transcriptome analysis of all major cell types within the human retina showed that TMEM97 is expressed in the RPE, retinal ganglion cells (RGCs) and amacrine cells. Using CRISPRi, we performed loss-of-function study of TMEM97 in the human RPE cell line, ARPE19. We generated a stable ARPE19 cell line expressing dCas9-KRAB which facilitated knockdown of TMEM97 using specific sgRNAs. Our results show that knockdown of TMEM97 in ARPE19 exerts a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports the role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi cell line generated here provided an useful in vitro tool for functional studies of other AMD-associated genes.

Stem cell and cell reprogramming technology represent a rapidly growing field in regenerative medicine. A number of novel neural reprogramming methods have been established, using pluripotent stem cells (PSCs) or direct reprogramming, to efficiently derive specific neuronal cell types for therapeutic applications. Both in vitro and in vivo cellular reprogramming provide diverse therapeutic pathways for modeling neurological diseases and injury repair. In particular, the retina has emerged as a promising target for clinical application of regenerative medicine. Herein, we review the potential of neuronal reprogramming to develop regenerative strategy, with a particular focus on treating retinal degenerative diseases and discuss future directions and challenges in the field.

Background Mesenchymal stem cells (MSCs) hold great potential as a therapy for stroke and have previously been shown to promote recovery in preclinical models of cerebral ischaemia. MSCs secrete a wide range of growth factors, chemokines, cytokines and extracellular vesicles—collectively termed the secretome. In this study, we assessed for the first time the efficacy of the IL-1α-primed MSC-derived secretome on brain injury and functional recovery after cerebral ischaemia. Methods Stroke was induced in male C57BL/6 mice using the intraluminal filament model of middle cerebral artery occlusion. Conditioned medium from IL-1α-primed MSCs or vehicle was administered at the time of reperfusion or at 24 h post-stroke by subcutaneous injection. Results IL-1α-primed MSC-derived conditioned medium treatment at the time of stroke led to a ~ 30% reduction in lesion volume at 48 h and was associated with modest improvements in body mass gain, 28-point neurological score and nest building. Administration of MSC-derived conditioned medium at 24 h post-stroke led to improved nest building and neurological score despite no observed differences in lesion volume at day 2 post-stroke. Conclusions Our results show for the first time that the administration of conditioned medium from IL-1α-primed MSCs leads to improvements in behavioural outcomes independently of neuroprotection.

Restoration of cerebral blood flow (CBF) and upregulation of angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Here, we investigated for the first time the role of PTX3 in long-term CBF, angiogenesis, and neuronal viability after ischaemic stroke induced by transient middle cerebral artery occlusion (MCAo). Lack of PTX3 had no effect on early brain damage, but significantly impaired restoration of CBF, 14 and 28 days after MCAo, compared to wild-type (WT) mice. Immunohistochemical analysis revealed that PTX3 KO mice have significantly greater neuronal loss, significantly decreased vessel diameter, vessel proliferation, vascular density, and reactive astrocytes and decreased expression of vascular endothelial growth factor receptor 2 (VEGR2), vascular extracellular matrix (ECM)-proteins (collagen IV, laminin), and integrin-β, in the ipsilateral (stroke) hemisphere compared to WT mice, 28 days after MCAo. Therefore, PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia. Collectively, these findings demonstrate the potential and clinical relevance of PTX3 as a promising therapeutic target, providing sustained long-term post-stroke neurovascular repair and reducing the loss of neurons. Key messages Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke.Restoration of cerebral blood flow (CBF) and angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia.PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia. Electronic supplementary material The online version of this article (10.1007/s00109-018-1698-6) contains supplementary material, which is available to authorized users.

Pluripotent 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. With the merging of modern technology and stem cell biology, an increased demand and implementation of automated platforms for stem cell studies is anticipated.

Surface topography can have significant impact on the cell fate decisions of stem cells. Construction of a biomaterial that can mimic the physiological microenvironment can be used to enhance the maintenance or differentiation of stem cells. Here, we manufacture two surfaces with monolayer binary colloidal crystals (BCCs) having different topographies, Si2PM and Si5PM, and examined their effect on maintenance and differentiation of human embryonic stem cells (hESCs). Our results demonstrated that BCCs can be coupled with vitronectin to maintain pluripotency of hESCs during culture, and with a gelatin coating to improve early definitive endoderm differentiation of hESCs using an embryonic body assay (i.e., FOXA2 up-regulation). BCCs can be used as novel cell culture substrates to support stem cell maintenance and differentiation.

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Patient-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. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60-positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium- to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

The revolution of induced pluripotent stem cell (iPSC) technology provides a platform for development of cell therapy, disease modeling and drug discovery. Recent technological advances now allow us to reprogram a patient's somatic cells into induced pluripotent stem cells (iPSCs). Together with methods to differentiate these iPSCs into disease-relevant cell types, we are now able to model disease in vitro using iPSCs. Importantly, this represents a robust in vitro platform using patient-specific cells, providing opportunity for personalized precision medicine. Here we provide a review of advances using iPSC for drug development, and discuss the potential and limitations of iPSCs for drug discovery in neurodegenerative and ocular diseases. Emerging technologies that can facilitate the search for new drugs by assessment using in vitro disease models will also be discussed, including organoid differentiation, organ-on-chip, direct reprogramming and humanized animal models.

Surface nanotopographies are an important way of mimicking the stem cell niche on biomaterial surfaces. Previous studies have focused on the differentiation of stem cells into a defined lineage using nanotopographies, but they have rarely considered the homogeneity of cell populations produced. We examined the impact of two types of substrates (i.e. nanogrooves and nanopillars made by soft lithography) on the surface-induced differentiation of human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) and mouse embryonic stem cells (mESCs) without the use of additional chemical induction medium components. Cell morphology and proliferation were analysed at day 1 and day 3. Gene expression was analysed at day 14 for hAM-MSCs and at day 7 for mESC-derived embryoid bodies (mEBs) using quantitative real-time polymerase chain reaction (qPCR). The substrates with nanogrooves had a noticeable effect on cell alignment in a depth dependent manner with both cell types showing strong alignment along the deep grooves. On the other hand, the nanopillar substrates showed inhibition of cell spreading for both cell types. The nanogrooves showed inhibition of hAM-MSC growth but enhanced mEB proliferation, especially on the deeper grooves. The nanopillars did not significantly affect hAM-MSC growth, but can modulate mEB growth depending on the pillar density, indicating that mEBs are more sensitive to nanotopographies in terms of proliferation, while hAM-MSCs are only sensitive to specific structures and sizes. Genes associated with bone, cartilage, and fat were investigated for hAM-MSCs, whereas genes of the endoderm, mesoderm, ectoderm, and pluripotency were investigated for mEBs. In general, gene expression for hAM-MSCs was not enhanced significantly by the nanotopographies compared to the flat control. On the other hand, genes of bone, cartilage, skeletal muscle, heart, and liver were up-regulated on both nanopillars and nanogrooves, especially OP65 (ordered pillars with 65% density) and SG40 (shallow grooves with 40 nm depth) in a feature size dependent manner. We found that a small portion of mEBs was composed of cardiac-like beating cells (i.e. GFP-NKX2.5 positive) and a bone cell marker (i.e. OCN) indicating a heterogeneous cell population being generated on those types of surfaces. This work highlights the importance of nanotopographies in stem cell differentiation and how studying multiple properties of the substrate and cells is needed as we strive to generate homogeneous and mature cell populations using biomaterials.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.

Ischemic stroke is a leading cause of death and long‐term disability worldwide, yet availability of current treatments is limited. The downstream events resulting from cerebral ischemia lead to an imbalance in the production of harmful reactive oxygen species (ROS) over endogenous antioxidant mechanisms. Thus, treatments that can reduce this imbalance can limit the extent of injury resulting from ischemic stroke. In this work, the potential of novel ROS‐scavenging PEGylated polymeric nanoparticles (NPs) composed of poly(propylene sulfide) (PPS) (PPS‐NPs) for ischemic stroke therapy is evaluated in vitro and in a mouse model of stroke. In vitro results show that PPS‐NPs display remarkable anti‐oxidant and anti‐inflammatory properties, whilst exhibiting negligible cytotoxicity. NPs administered intravenously rapidly accumulate in ischemic brain regions as visualized through fluorescence imaging, reduced infarct volume, blood‐brain barrier damage, neuronal loss, and neuroinflammation as determined by immunohistochemistry, and improved recovery of neurological function as determined through behavioral analyses. Crucially, the data show that the therapeutic time window for administration of PPS‐NPs extends up to 3 h, a clinically relevant time window for stroke. The findings provide new strong evidence that these NPs may be an effective antioxidant therapy for ischemic stroke.

The development of new therapies for stroke continues to face repeated translational failures. Brain endothelial cells form paracellular and transcellular barriers to many blood-borne therapies and the development of efficient delivery strategies is highly warranted. Here, in a mouse model of stroke, we show selective recruitment of clinically used liposomes into the ischaemic brain that correlates with biphasic blood brain barrier (BBB) breakdown. Intravenous administration of liposomes into mice exposed to transient middle cerebral artery occlusion took place at early (0.5h and 4h) and delayed (24h and 48h) timepoints, covering different phases of BBB disruption after stroke. Using a combination of in vivo real-time imaging and histological analysis we show that selective liposomal brain accumulation coincides with biphasic enhancement in transcellular transport followed by a delayed impairment to the paracellular barrier. This process precedes neurological damage in the acute phase and maintains long-term liposomal co-localisation within the neurovascular unit, which could have great potential for neuroprotection. Levels of liposomal uptake by glial cells are similarly selectively enhanced in the ischaemic region late after experimental stroke (2-3 days), highlighting their potential for blocking delayed inflammatory responses or shifting the polarization of microglia/macrophages towards brain repair.These findings demonstrate the capability of liposomes to maximise selective translocation into the brain after stroke and identify for the first time two windows for therapeutic manipulation. This emphasizes the benefits of selective drug delivery for efficient tailoring of new stroke treatments.

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that β1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. β1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and β1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that β1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, β1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis. This article has an associated First Person interview with the first author of the paper., Summary: By using flow systems, magnetic tweezers and knockout mice, we show that β1 integrin is activated by unidirectional hemodynamic shear force leading to Ca2+ signalling and cell alignment, but it is not activated by bidirectional force.

Background Stroke remains a leading cause of death and disability worldwide despite recent treatment breakthroughs. A primary event in stroke pathogenesis is the development of a potent and deleterious local and peripheral inflammatory response regulated by the pro-inflammatory cytokine interleukin-1 (IL-1). While the role of IL-1β (main released isoform) has been well studied in stroke, the role of the IL-1α isoform remains largely unknown. With increasing utilization of intravenous tissue plasminogen activator (t-PA) or thrombectomy to pharmacologically or mechanically remove ischemic stroke causing blood clots, respectively, there is interest in pairing successful cerebrovascular recanalization with neurotherapeutic pharmacological interventions (Fraser et al., J Cereb Blood Flow Metab 37:3531–3543, 2017; Hill et al., Lancet Neurol 11:942–950, 2012; Amaro et al., Stroke 47:2874–2876, 2016). Methods Transient stroke was induced in mice via one of two methods. One group of mice were subjected to tandem ipsilateral common carotid artery and middle cerebral artery occlusion, while another group underwent the filament-based middle cerebral artery occlusion. We have recently developed an animal model of intra-arterial (IA) drug administration after recanalization (Maniskas et al., J Neurosci Met 240:22–27, 2015). Sub groups of the mice were treated with either saline or Il-1α, wherein the drug was administered either acutely (immediately after surgery) or subacutely (on the third day after stroke). This was followed by behavioral and histological analyses. Results We now show in the above-mentioned mouse stroke models (transient tandem ipsilateral common carotid artery (CCA) and middle cerebral artery occlusion (MCA) occlusion, MCA suture occlusion) that IL-1α is neuroprotective when acutely given either intravenously (IV) or IA at low sub-pathologic doses. Furthermore, while IV administration induces transient hemodynamic side effects without affecting systemic markers of inflammation, IA delivery further improves overall outcomes while eliminating these side effects. Additionally, we show that delayed/subacute IV IL-1α administration ameliorates functional deficit and promotes neurorepair. Conclusions Taken together, our present study suggests for the first time that IL-1α could, unexpectedly, be an effective ischemic stroke therapy with a broad therapeutic window.

Reprogramming 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. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.

Aims We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 ( ALPK3 ) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. Methods and results We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. Conclusions This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo . Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.

We 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.

Background: Following ischemic stroke, recanalisation and restoration of blood flow to the affected area of the brain is critical and directly correlates with patient recovery. In vivo models of ischemic stroke show high variability in outcomes, which may be due to variability in reperfusion. We previously reported that a surgical refinement in the middle cerebral artery occlusion (MCAO) model of stroke, via repair of the common carotid artery (CCA), removes the reliance on the Circle of Willis for reperfusion and reduced infarct variability. Here we further assess this refined surgical approach on reperfusion characteristics following transient MCAO in mice. Methods: Mice underwent 60 min of MCAO, followed by either CCA repair or ligation at reperfusion. All mice underwent laser speckle contrast imaging at baseline, 24 h and 48 h post-MCAO. Results: CCA ligation reduced cerebral perfusion in the ipsilateral hemisphere compared to baseline (102.3 ± 4.57%) at 24 h (85.13 ± 16.09%; P < 0.01) and 48 h (75.04 ± 12.954%; P < 0.001) post-MCAO. Repair of the CCA returned perfusion to baseline (94.152 ± 2.44%) levels and perfusion was significantly improved compared to CCA ligation at both 24 h (102.83 ± 8.41%; P < 0.05) and 48 h (102.13 ± 9.34%; P < 0.001) post-MCAO. Conclusions: Our findings show CCA repair, an alternative surgical approach for MCAO, results in improved ischemic hemisphere perfusion during the acute phase.

BACKGROUND: Home oxygen therapy is commonly prescribed for patients who remain hypoxemic at hospital discharge, although evidence supporting this practice is lacking. This study aimed to evaluate oxygen prescription and follow-up for patients who were prescribed post-discharge short-term oxygen therapy (STOT) and to assess their long-term outcome. METHODS: A retrospective audit was undertaken of subjects prescribed STOT following hospitalization at a single site in Melbourne, Australia, between January 2011 and December 2015. During the study period, a designated clinical pathway for STOT prescription and follow-up after hospital discharge was in place. Chart review was performed to collect subject demographics and comorbidities, results of oxygen assessment (arterial blood gas and 6-min walk tests) and prescription, and results at follow-up re-assessment and mortality. RESULTS: Over five 5 years, 205 subjects were prescribed STOT upon hospital discharge. Common indications for oxygen treatment were chronic lung disease (54%) and dyspnea palliation (26%). Of the 152 subjects who were discharged with non-palliative oxygen therapy, 28% did not fulfil the recommended prescribing criteria or did not have recommended assessments. Among the 118 subjects who returned for re-assessment 4 weeks after initial oxygen provision, 47 (40%) did not fulfill criteria for long-term oxygen therapy. The 1-y cumulative survival rate for the study population was 56%. CONCLUSIONS: A significant proportion of subjects who were prescribed post-discharge STOT did not fulfill the recommended prescribing criteria. The long-term prognosis for subjects who were prescribed post-discharge STOT was poor.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Muller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Muller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

SummaryThe retina is a highly specialized neural tissue that senses light and initiates image processing. Although the functional organisation of specific cells within the retina has been well-studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile cell types in the human retina, we performed single cell RNA-sequencing on 20,009 cells obtained post-mortem from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct clusters representing all known retinal cells: rod photoreceptors, cone photoreceptors, Müller glia cells, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, retinal astrocytes and microglia. Notably, our data captured molecular profiles for healthy and early degenerating rod photoreceptors, and revealed a novel role of MALAT1 in putative rod degeneration. We also demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to our understanding of retinal biology and disease.

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here we report a simple, cloning-free method to induce gene expression at endogenous locus using CRISPR/Cas9 activators. Our strategy utilises synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65 and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this cloning-free approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1 and NEUROD1) in human cells, whereas the rat cells exhibit a more variable and less efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applies as a rapid workflow to initiate gain-of-function studies for a range of molecular and cell biology disciplines.

Endovascular thrombectomy combined with t-PA is the current standard of care for emergent large vessel occlusion (ELVO) stroke. Unfortunately, despite rising recanalization rates, stroke remains the leading cause of long-term disability worldwide suggesting that additional therapies are needed. Severe stroke morbidity may be due, partially, to the acute and sustained inflammatory stroke response. Preclinical research has shown some promise with anti-inflammatory agents in limiting brain injury and improving functional outcome; however, the post-stroke inflammatory cascade appears to have both beneficial and deleterious effects making the translation of such anti-inflammatory approaches perilous. We aim to show that the inflammatory cytokine interleukin-1alpha (IL-1α) is well-tolerated, neuroprotective, and conveys functional benefit after ischemic stroke. 3-month-old C57/BL6 mice were subjected to MCAo and given IL-1α via IV injection or IA infusion upon recanalization. Mice were allowed to recover for 7 days before sacrifice. Brains were flash frozen for IHC and serum for high sensitivity ELISA. We noted that IV IL-1α (1 ng) is neuroprotective (as measured by cresyl violet stained infarct volumes) and significantly improved functional recovery in open field behavioral tests. When given IV, IL-1α showed transient hemodynamic side effects that are typical of IL-1α’s known physiologic functions (fever, sterile inflammation, etc.) These effects are short lived with systemic (IV) administration and IA IL-1α (0.1 ng) administration showed neuroprotection without the side effects seen with IV treatment. Additionally, we noted that IL-1α is directly neuroprotective of primary mouse cortical neurons exposed to oxygen and glucose deprivation conditions in vitro . IL-1α’s profound, direct neuroprotective effects and its functional benefit, make IL-1α an attractive target for future study in ischemic stroke.

Despite recent advancements in blood vessel recanalization, ischemic stroke remains a leading cause of significant morbidity. Therefore, new stroke therapies are urgently needed, but have thus far failed in clinical trials despite promising animal data. Studies in our lab have suggested that proteolytic fragments of the extracellular matrix heparan sulfate proteoglycan perlecan are neuroprotective and neuroreparative in experimental ischemic stroke. Further studies have suggested that one such fragment, perlecan LG3, is generated by multiple cells of the neurovascular unit in response to the pro-inflammatory cytokine, interleukin-1alpha (IL-1α). Long thought to be deleterious to the brain after stroke, we now provide data that suggests that IL-1α is neuroreparative upon delayed intravenous administration in experimental ischemic stroke. Coupled with other work in our lab that suggests that acute post-stroke IL-1α administration is neuroprotective, we here aim to show that IL-1α is also an attractive therapeutic target in later phases of stroke. 3-month-old C57Bl6 mice underwent transient middle cerebral artery occlusion for one hour and received either vehicle or IL-1α treatment (1ng/mouse) intravenously 3 days following experimental stroke. For up to 14 days after stroke, all groups were evaluated on an 11 point neuroscore. Mice were sacrificed 7 or 14 days post stroke and their brains analyzed for cellular dysmorphia, endothelial cell activation, overall cerebral vascularization, and evidence of neurogenesis at the subventricular zone (SVZ). IL-1α treated mice have less brain dysmorphia (p > 0.0001), greater overall vascularization (p > 0.05), a trending increase in neurogenesis at the SVZ, and significantly improved behavioral outcomes. Collectively, our results suggest that IL-1α could represent an unexpected and novel therapeutic approach for ischemic stroke with a broad therapeutic window.

The San Francisco and Lisbon estuaries share many geographical similarities, but their different governance makes for interesting comparisons. Many tributaries to San Francisco Bay were channelized by the US Army Corps in the 1950s–1970s. The design flaws of these projects (such as their having ignored sediment) are manifest as local governments now struggle to maintain and operate them. Local agencies in the San Francisco region have used a range of innovative tools to solve these flood risk problems, such as creation of a new governance structure encompassing multiple jurisdictions around a stream, adoption of a 50-year plan to convert aging concrete channels into natural creeks, and implementation of projects that combine flood risk reduction with ecosystem and social benefits. Such a multi-purpose project built on the Ribeira das Jardas Stream near Lisbon has proven highly successful, especially as an urban social space.

Stroke represents a global challenge and is a leading cause of permanent disability worldwide. Despite much effort, translation of research findings to clinical benefit has not yet been successful. Failure of neuroprotection trials is considered, in part, due to the low quality of preclinical studies, low level of reproducibility across different laboratories and that stroke co-morbidities have not been fully considered in experimental models. More rigorous testing of new drug candidates in different experimental models of stroke and initiation of preclinical cross-laboratory studies have been suggested as ways to improve translation. However, to our knowledge, no drugs currently in clinical stroke trials have been investigated in preclinical cross-laboratory studies. The cytokine interleukin 1 (IL-1) is a key mediator of neuronal injury and the naturally occurring IL-1 receptor antagonist (IL-1Ra) has been reported as beneficial in experimental studies of stroke. In the present paper, we report on a preclinical cross-laboratory stroke trial designed to investigate the efficacy of IL-1Ra in different research laboratories across Europe. Our results strongly support the therapeutic potential of IL-1Ra in experimental stroke and provide further evidence that IL-1Ra should be evaluated in more extensive clinical stroke trials.

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby's fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich "drusen-like" composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE-ECM interface.

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information)., The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Meetings of the working group were funded by the NC3Rs. Dr. Michael J. O Neill is a full time employee of Eli Lilly & Co.

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.