Automated image analysis system for studying cardiotoxicity in human pluripotent stem cell-Derived cardiomyocytes

BackgroundCardiotoxicity, characterized by severe cardiac dysfunction, is a major problem in patients treated with different classes of anticancer drugs. Development of predictable human-based models and assays for drug screening are crucial for preventing potential drug-induced adverse effects. Current animal in vivo models and cell lines are not always adequate to represent human biology. Alternatively, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show great potential for disease modelling and drug-induced toxicity screenings. Fully automated high-throughput screening of drug toxicity on hiPSC-CMs by fluorescence image analysis is, however, very challenging, due to clustered cell growth patterns and strong intracellular and intercellular variation in the expression of fluorescent markers.ResultsIn this paper, we report on the development of a fully automated image analysis system for quantification of cardiotoxic phenotypes from hiPSC-CMs that are treated with various concentrations of anticancer drugs doxorubicin or crizotinib. This high-throughput system relies on single-cell segmentation by nuclear signal extraction, fuzzy C-mean clustering of cardiac α-actinin signal, and finally nuclear signal propagation. When compared to manual segmentation, it generates precision and recall scores of 0.81 and 0.93, respectively.ConclusionsOur results show that our fully automated image analysis system can reliably segment cardiomyocytes even with heterogeneous α-actinin signals.Computer Systems, Imagery and MediaAlgorithms and the Foundations of Software technolog

Embedded macrophages induce intravascular coagulation in 3D blood vessel-on-chip

Macrophages are innate immune cells that prevent infections and help in wound healing and vascular inflammation. While these cells are natural helper cells, they also contribute to chronic diseases, e.g., by infiltrating the endothelial layer in early atherosclerosis and by promoting vascular inflammation. There is a crosstalk between inflammatory pathways and key players in thrombosis, such as platelets and endothelial cells – a phenomenon known as ‘thromboinflammation’. The role of the embedded macrophages in thromboinflammation in the context of vascular disease is incompletely understood. Blood vessels-on-chips, which are microfluidic vascular cell culture models, have been used extensively to study aspects of vascular disease, like permeability, immune cell adhesion and thrombosis. Blood perfusion assays in blood vessel-on-chip models benefit from multiple unique aspects of the models, such as control of microvessel structure and well-defined flow patterns, as well as the ability to perform live imaging. However, due to their simplified nature, blood vessels-on-chip models have not yet been used to capture the complex cellular crosstalk that is important in thromboinflammation. Using induced pluripotent stem cell-derived endothelial cells and polarized THP-1 monocytes, we have developed and systematically set up a 3D blood vessel-on-chip with embedded (lipid-laden) macrophages, which is created using sequential cell seeding in viscous finger patterned collagen hydrogels. We have set up a human whole blood perfusion assay for these 3D blood vessels-on-chip. An increased deposition of fibrin in the blood vessel-on-chip models containing lipid-laden macrophages was observed. We anticipate the future use of this advanced vascular in vitro model in drug development for early atherosclerosis or aspects of other vascular diseases

The impact of cannabidiol treatment on resting state functional connectivity, prefrontal metabolite levels and reward processing in recent-onset patients with a psychotic disorder

The first clinical trials with cannabidiol (CBD) as treatment for psychotic disorders have shown its potential as an effective and well-tolerated antipsychotic agent. However, the neurobiological mechanisms underlying the antipsychotic profile of CBD are currently unclear. Here we investigated the impact of 28-day adjunctive CBD or placebo treatment (600 mg daily) on brain function and metabolism in 31 stable recent-onset psychosis patients (&lt;5 years after diagnosis). Before and after treatment, patients underwent a Magnetic Resonance Imaging (MRI) session including resting state functional MRI, proton Magnetic Resonance Spectroscopy (1H-MRS) and functional MRI during reward processing. Symptomatology and cognitive functioning were also assessed. CBD treatment significantly changed functional connectivity in the default mode network (DMN; time × treatment interaction p = 0.037), with increased connectivity in the CBD (from 0.59 ± 0.39 to 0.80 ± 0.32) and reduced connectivity in the placebo group (from 0.77 ± 0.37 to 0.62 ± 0.33). Although there were no significant treatment effects on prefrontal metabolite concentrations, we showed that decreased positive symptom severity over time was associated with both diminishing glutamate (p = 0.029) and N-acetyl-aspartate (NAA; neuronal integrity marker) levels (p = 0.019) in the CBD, but not the placebo group. CBD treatment did not have an impact on brain activity patterns during reward anticipation and receipt or functional connectivity in executive and salience networks. Our results show that adjunctive CBD treatment of recent-onset psychosis patients induced changes in DMN functional connectivity, but not prefrontal metabolite concentrations or brain activity during reward processing. These findings suggest that DMN connectivity alteration may be involved in the therapeutic effects of CBD.</p

Multiplexed fluidic circuit board for controlled perfusion of 3D blood vessels-on-a-chip

Three-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and circumferential strain (CS). However, these parameters are difficult to control and are often poorly reproducible due to the high intrinsic diameter variation of individual 3D-VoCs. As a result, the throughput of current 3D systems is one-channel-at-a-time. Here, we developed a fluidic circuit board (FCB) for simultaneous perfusion of up to twelve 3D-VoCs using a single set of control parameters. By designing the internal hydraulic resistances in the FCB appropriately, it was possible to provide a pre-set WSS to all connected 3D-VoCs, despite significant variation in lumen diameters. Using this FCB, we found that variation of CS or WSS induce morphological changes to human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and conclude that control of these parameters using a FCB is necessary to study 3D-VOCs.Stem cells & developmental biolog

Allocation to highly sensitized patients based on acceptable mismatches results in low rejection rates comparable to nonsensitized patients

Contains fulltext : 208426.pdf (publisher's version ) (Open Access)Whereas regular allocation avoids unacceptable mismatches on the donor organ, allocation to highly sensitized patients within the Eurotransplant Acceptable Mismatch (AM) program is based on the patient's HLA phenotype plus acceptable antigens. These are HLA antigens to which the patient never made antibodies, as determined by extensive laboratory testing. AM patients have superior long-term graft survival compared with highly sensitized patients in regular allocation. Here, we questioned whether the AM program also results in lower rejection rates. From the PROCARE cohort, consisting of all Dutch kidney transplants in 1995-2005, we selected deceased donor single transplants with a minimum of 1 HLA mismatch and determined the cumulative 6-month rejection incidence for patients in AM or regular allocation. Additionally, we determined the effect of minimal matching criteria of 1 HLA-B plus 1 HLA-DR, or 2 HLA-DR antigens on rejection incidence. AM patients showed significantly lower rejection rates than highly immunized patients in regular allocation, comparable to nonsensitized patients, independent of other risk factors for rejection. In contrast to highly sensitized patients in regular allocation, minimal matching criteria did not affect rejection rates in AM patients. Allocation based on acceptable antigens leads to relatively low-risk transplants for highly sensitized patients with rejection rates similar to those of nonimmunized individuals