iPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype.

BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.

Three-dimensional bioprinting of stem cell-derived central nervous system cells enables astrocyte growth, vasculogenesis, and enhances neural differentiation/function.

Current research tools for preclinical drug development such as rodent models and two-dimensional immortalized monocultures have failed to serve as effective translational models for human central nervous system (CNS) disorders. Recent advancements in the development of induced pluripotent stem cells (iPSCs) and three-dimensional (3D) culturing can improve the in vivo-relevance of preclinical models, while generating 3D cultures though novel bioprinting technologies can offer increased scalability and replicability. As such, there is a need to develop platforms that combine iPSC-derived cells with 3D bioprinting to produce scalable, tunable, and biomimetic cultures for preclinical drug discovery applications. We report a biocompatible poly(ethylene glycol)-based matrix which incorporates Arg-Gly-Asp and Tyr-Ile-Gly-Ser-Arg peptide motifs and full-length collagen IV at a stiffness similar to the human brain (1.5 kPa). Using a high-throughput commercial bioprinter we report the viable culture and morphological development of monocultured iPSC-derived astrocytes, brain microvascular endothelial-like cells, neural progenitors, and neurons in our novel matrix. We also show that this system supports endothelial-like vasculogenesis and enhances neural differentiation and spontaneous activity. This platform forms a foundation for more complex, multicellular models to facilitate high-throughput translational drug discovery for CNS disorders.

Perphenazine-Macrocycle Conjugates Rapidly Sequester the Aß42 Monomer and Prevent Formation of Toxic Oligomers and Amyloid.

Alzheimer's disease is imposing a growing social and economic burden worldwide, and effective therapies are urgently required. One possible approach to modulation of the disease outcome is to use small molecules to limit the conversion of monomeric amyloid (Aß42) to cytotoxic amyloid oligomers and fibrils. We have synthesized modulators of amyloid assembly that are unlike others studied to date: these compounds act primarily by sequestering the Aß42 monomer. We provide kinetic and nuclear magnetic resonance data showing that these perphenazine conjugates divert the Aß42 monomer into amorphous aggregates that are not cytotoxic. Rapid monomer sequestration by the compounds reduces fibril assembly, even in the presence of pre-formed fibrillar seeds. The compounds are therefore also able to disrupt monomer-dependent secondary nucleation, the autocatalytic process that generates the majority of toxic oligomers. The inhibitors have a modular design that is easily varied, aiding future exploration and use of these tools to probe the impact of distinct Aß42 species populated during amyloid assembly.

Deep vein thrombosis: diagnosis of a comon clinical problem.

Ultrasound is recognized as a very accurate first line diagnostic tool when a patient presents with a symptomatic lower extremity suggesting acute venous thrombus. Advantages of ultrasound are that it does not involve using radiation or contrast and can be performed portably, generally with no complications. In many patients, other abnormalities such as Baker's cysts, arterial aneurysms, or hematomas can be detected and can explain the symptoms. Some limitations are that ultrasound is dependent on the skills of the sonographer and is very limited in visualization of iliac and abdominal veins. We are currently investigating computed tomography (CT) venography, which appears to be an accurate alternative, particularly for obese patients or when pelvic or abdominal thrombus is suspected.

Pulmonary thromboembolic disease: a new role for computed tomography.

Over the past few years, computed tomography (CT) has emerged as a common noninvasive, definitive, alternative to ventilation-perfusion scintigraphy scan and pulmonary angiography in the evaluation of patients suspected of having pulmonary emboli. Additionally, recent articles have investigated the possibility of using CT to identify deep venous thrombi following a spiral CT pulmonary angiogram. Using the same bolus of contrast as that administered for a CT pulmonary angiogram, the ultimate goal is to design a single test that defines both aspects of pulmonary thromboembolic disease. More studies are needed and controversy exists, but CT's role in the evaluation of pulmonary thromboembolic disease appears promising.