Biology subjects

Murray, P.

Publications and source records attributed to Murray, P..

8 recordsLinked to original sources

bioRxiv2018

In vivo fate of free and encapsulated iron oxide nanoparticles after injection of labelled stem cells

Nanoparticle contrast agents are useful tools to label stem cells and monitor the in vivo bio-distribution of labeled cells in pre-clinical models of disease. In this context, understanding the in vivo fate of the particles after injection of labelled cells is important for their eventual clinical use as well as for the interpretation of imaging results. We examined how the formulation of superparamagnetic iron oxide nanoparticles (SPIONs) impacts the labelling efficiency, magnetic characteristics and fate of the particles by comparing individual SPIONs with polyelectrolyte multilayer capsules containing SPIONs. At low labelling concentration, encapsulated SPIONs served as an efficient labelling agent for stem cells. The bio-distribution after intra-cardiac injection of labelled cells was monitored longitudinally by MRI and as an endpoint by inductively coupled plasma-optical emission spectrometry. The results suggest that, after being released from labelled cells after cell death, both formulations of particles are initially stored in liver and spleen and are not completely cleared from these organs 2 weeks post-injection.

biochemistry↗

bioRxiv2018

Development of an imaging toolbox to assess the therapeutic potential and biodistribution of macrophages in a mouse model of multiple organ dysfunction

Cell-based regenerative medicine therapies require robust preclinical safety, efficacy, biodistribution and engraftment data prior to clinical testing. To address these challenges, we have developed an imaging toolbox comprising multi-spectral optoacoustic tomography and ultrasonography, which allows the degree of kidney, liver and cardiac injury and the extent of functional recovery to be assessed non-invasively in a mouse model of multi-organ dysfunction. This toolbox allowed us to determine the therapeutic effects of adoptively transferred M2 macrophages. Using bioluminescence imaging, we could then investigate the association between amelioration and biodistribution. Macrophage therapy improved kidney and liver function to a limited extent, but did not ameliorate histological damage. No improvement in cardiac function was observed. Biodistribution analysis showed that macrophages homed and persisted in the injured kidneys and liver, but did not populate the heart. Our data suggest that the limited improvement observed in kidney and liver function could be mediated by M2 macrophages.

pharmacology and toxicology↗

bioRxiv2018

CDK1 and CDK2 regulate phosphorylation-dependent NICD1 turnover and the periodicity of the segmentation clock

All vertebrates share a segmented body axis. Segments form periodically from the rostral end of the presomitic mesoderm (PSM) and this periodicity is regulated by the segmentation clock, a molecular oscillator that drives dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanistic details of how NICD stability is regulated are unclear.\n\nWe identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle-dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations.

developmental biology↗

bioRxiv2017

Comparing the differentiation potential of Brachyury+ mesodermal cells generated from 3-D and 2-D culture systems

Mesodermal populations can be generated in vitro from mouse embryonic stem cells (mESCs) using three-dimensional (3-D) aggregates called embryoid bodies or two-dimensional (2-D) monolayer culture systems. Here, we investigated whether Brachyury-expressing mesodermal cells generated using 3-D or 2-D culture systems are equivalent, or instead, have different properties. Using a Brachyury-GFP/E2-Crimson reporter mESC line, we isolated Brachyury-GFP+ mesoderm cells using flow-activated cell sorting and compared their gene expression profiles and ex vivo differentiation patterns. Quantitative RT-PCR analysis showed significant up-regulation of Cdx2, Foxf1 and Hoxb1 in the Brachyury-GFP+ cells isolated from the 3-D system compared with those isolated from the 2-D system. Furthermore, using an ex vivo mouse kidney rudiment assay, we found that irrespective of their source, Brachyury-GFP+ cells failed to integrate into developing nephrons, which are derived from the intermediate mesoderm. However, Brachyury-GFP+ cells isolated under 3-D conditions appeared to differentiate into endothelial-like cells within the kidney rudiments, whereas the Brachyury-GFP+ isolated from the 2-D conditions only did so to a limited degree. The high expression of Foxf1 in the 3-D Brachyury-GFP+ cells combined with their tendency to differentiate into endothelial-like cells suggests these mesodermal cells may represent lateral plate mesoderm.

developmental biology↗

bioRxiv2017

Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy

Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregation in vitro assays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect development in vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.

developmental biology↗

bioRxiv2017

Magnetic resonance imaging for characterization of a chick embryo model of cancer cell metastases

BackgroundMetastasis is the most common cause of death for cancer patients, hence its study has rapidly expanded over the past few years. To fully understand all the steps involved in metastatic dissemination, in vivo models are required, of which murine ones are the most common. Therefore pre-clinical imaging methods have mainly been developed for small mammals. However, the potential of preclinical imaging techniques such as magnetic resonance imaging (MRI) to monitor cancer growth and metastasis in non-mammalian in vivo models is not commonly used. We have here used MRI to measure primary neuroblastoma tumour size and presence of metastatic dissemination in a chick embryo model. We compared its sensitivity and accuracy to end-point fluorescence detection.\n\nMethodsHuman neuroblastoma cells were labelled with GFP and micron-sized iron particles (MPIOs) and implanted on the extraembryonic chorioallantoic membrane of the chick embryo at E7. T2 RARE, T2 weighted FLASH as well as time-of-flight MR angiography imaging was applied at E14. Primary tumours as well as metastatic deposits in the chick embryo were dissected post imaging to compare with MRI results.\n\nResultsMPIO labelling of neuroblastoma cells allowed in ovo observation of the primary tumour and tumour volume measurement non-invasively over time. Moreover, T2 weighted and FLASH imaging permitted the detection of very small metastatic deposits in the chick embryo.\n\nConclusionsThe use of contrast agents enabled the detection of metastatic deposits of neuroblastoma cells in a chick embryo model, thereby reinforcing the potential of this cost efficient and convenient, 3R compliant, in vivo model for cancer research.

cancer biology↗

bioRxiv2017

Non-invasive imaging platform reveals a potential tumourigenicity hazard of systemically administered cells

BackgroundCell-based regenerative medicine therapies are now frequently tested in clinical trials. In many conditions, cell therapies are administered systemically, but there is little understanding of their fate, and adverse events are often under-reported. Currently, it is only possible to assess safety and fate of cell therapies in preclinical studies, specifically by monitoring animals longitudinally using multimodal imaging approaches. Here, using a suite of in vivo imaging modalities to explore the fate of a range of human and murine cells, we investigate how route of administration, cell type and host immune status affect the fate of administered cells.\n\nMethodsWe applied a unique imaging toolkit combining bioluminescence, optoacoustic and magnetic resonance imaging modalities to assess the safety of different human and murine cell types by following their biodistribution and persistence in mice following administration into the venous or arterial system. Results: Longitudinal imaging analyses (i) suggested that the intra-arterial route may be more hazardous than intravenous administration for certain cell types; (ii) revealed that the potential of a mouse mesenchymal stem/stromal cell (MSC) line to form tumours, depended on administration route and mouse strain; and (iii) indicated that clinically tested human umbilical cord (hUC)-derived MSCs can transiently and unexpectedly proliferate when administered intravenously to mice.\n\nConclusionsIn order to perform an adequate safety assessment of potential cell-based therapies, a thorough understanding of cell biodistribution and fate post administration is required. The non-invasive imaging toolbox used here can expose not only the general organ distribution of these therapies, but also a detailed view of their presence within different organs and, importantly, tumourigenic potential. Our observation that the hUC-MSCs but not the human bone marrow (hBM)-derived MSCs persisted for a period in some animals, suggests that therapies with these cells should proceed with caution.

cell biology↗

bioRxiv2017

Multimodal cell tracking from systemic administration to tumour growth by combining gold nanorods and reporter genes

Understanding the fate of exogenous cells after implantation is important for clinical applications. Preclinical studies allow imaging of cell location and survival. Labelling with nanoparticles enables high sensitivity detection, but cell division and cell death cause signal dilution and false positives. By contrast, genetic reporter signals are amplified by cell division. Here we characterise lentivirus-based bi-cistronic reporter gene vectors and silica-coated gold nanorods (GNRs) as synergistic tools for cell labelling and tracking. Co-expression of the bioluminescence reporter luciferase and the optoacoustic reporter near-infrared fluorescent protein iRFP720 enabled cell tracking over time in mice. Multispectral optoacoustic tomography (MSOT) showed immediate biodistribution of GNR-labelled cells after intracardiac injection and successive clearance of GNRs (day 1-15) with high resolution, while optoacoustic iRFP720 detection indicated tumour growth (day 10-40). This multimodal cell tracking approach could be applied widely for cancer and regenerative medicine research to monitor short- and long-term biodistribution, tumour formation and metastasis.

cell biology↗