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

MS-1 magA: Revisiting Its Efficacy as a Reporter Gene for MRI

Bacterial genes involved in the biomineralization of magnetic nanoparticles in magnetotactic bacteria have recently been proposed as reporters for magnetic resonance imaging (MRI). In such systems, the expression of the bacterial genes in mammalian cells purportedly leads to greater concentrations of intracellular iron or the biomineralization of iron oxides, thus leading to an enhancement in relaxation rate that is detectable via MRI. Here, we show that the constitutive expression of the magA gene from Magnetospirillum magnetotacticum is tolerated by human embryonic kidney (HEK) cells but induces a strong toxic effect in murine mesenchymal/stromal cells and kidney-derived stem cells, severely restricting its effective use as a reporter gene for stem cells. Although it has been suggested that magA is involved in iron transport, when expressed in HEK cells, it does not affect the transcription of endogenous genes related to iron homeostasis. Furthermore, the magA -induced enhancement in iron uptake in HEK cells is insignificant, suggesting this gene is a poor reporter even for cell types that can tolerate its expression. We suggest that the use of magA for stem cells should be approached with caution, and its efficacy as a reporter gene requires a careful assessment on a cell-by-cell basis

Spatial epidemiological patterns suggest mechanisms of land-sea transmission for Sarcocystis neurona in a coastal marine mammal.

Sarcocystis neurona was recognised as an important cause of mortality in southern sea otters (Enhydra lutris nereis) after an outbreak in April 2004 and has since been detected in many marine mammal species in the Northeast Pacific Ocean. Risk of S. neurona exposure in sea otters is associated with consumption of clams and soft-sediment prey and is temporally associated with runoff events. We examined the spatial distribution of S. neurona exposure risk based on serum antibody testing and assessed risk factors for exposure in animals from California, Washington, British Columbia and Alaska. Significant spatial clustering of seropositive animals was observed in California and Washington, compared with British Columbia and Alaska. Adult males were at greatest risk for exposure to S. neurona, and there were strong associations with terrestrial features (wetlands, cropland, high human housing-unit density). In California, habitats containing soft sediment exhibited greater risk than hard substrate or kelp beds. Consuming a diet rich in clams was also associated with increased exposure risk. These findings suggest a transmission pathway analogous to that described for Toxoplasma gondii, with infectious stages traveling in freshwater runoff and being concentrated in particular locations by marine habitat features, ocean physical processes, and invertebrate bioconcentration

Plasma Polymer Coatings To Direct the Differentiation of Mouse Kidney-Derived Stem Cells into Podocyte and Proximal Tubule-like Cells

Kidney disease is now recognised as a global health problem and is associated with increased morbidity and mortality, along with high economic costs. To develop new treatments for ameliorating kidney injury and preventing disease progression, there is a need for appropriate renal culture systems for screening novel drugs and investigating the cellular mechanisms underlying renal pathogenesis. There is a need for in vitro culture systems that promote the growth and differentiation of specialised renal cell types. In this work, we have used plasma polymerisation technology to generate gradients of chemical functional groups to explore whether specific concentrations of these functional groups can direct the differentiation of mouse kidney-derived stem cells into specialised renal cell types. We found that amine-rich (-NH2) allylamine-based plasma polymerised coatings could promote differentiation into podocyte-like cells, whereas methyl-rich (CH3) 1,7-octadiene-based coatings promoted differentiation into proximal tubule-like cell (PTC). Importantly, the PT-like cells generated on the substrates expressed the marker megalin and were able to endocytose albumin, indicating that the cells were functional

Tailoring the surface charge of dextran-based polymer coated SPIONs for modulated stem cell uptake and MRI contrast

Tracking stem cells in vivo using non-invasive techniques is critical to evaluate the efficacy and safety of stem cell therapies. Superparamagnetic iron oxide nanoparticles (SPIONs) enable cells to be tracked using magnetic resonance imaging (MRI), but to obtain detectable signal cells need to be labelled with a sufficient amount of iron oxide. For the majority of SPIONs, this can only be obtained with the use of transfection agents, which can adversely affect cell health. Here, we have synthesised a library of dextran-based polymer coated SPIONs with varying surface charge from −1.5 mV to +18.2 mV via a co-precipitation approach and investigated their ability to be directly internalised by stem cells without the need for transfection agents. The SPIONs were colloidally stable in physiological solutions. The crystalline phase of the particles was confirmed with powder X-ray diffraction and their magnetic properties were characterised using SQUID magnetometry and magnetic resonance. Increased surface charge led to six-fold increase in uptake of particles into stem cells and higher MRI contrast, with negligible change in cell viability. Cell tracking velocimetry was shown to be a more accurate method for predicting MRI contrast of stem cells compared to measuring iron oxide uptake through conventional bulk iron quantification

Infant formula feeding practices in a prospective population based study

Background: It is recommended that formula-fed infants are given standard whey-based infant formula throughout the first year of life, unless otherwise advised by healthcare professionals. To our knowledge it has not yet been explored if parents are using a whey-based infant formula throughout the first 12 months of life. Reasons for parental choice of formula are also unknown. Therefore, the objective of this paper was to describe parental administration of whey-based and non whey-based infant formula in the first year of life. Methods: Data collected as part of the Cork BASELINE Birth Cohort Study examined infant feeding practices at 2, 6 and 12 months of age. Descriptive analysis explored infant feeding practices and parental reasons for changing from a whey-based to a non whey-based infant formula. Multiple logistic regression investigated parental and infant characteristics associated with the use of whey-based infant formula. Results: In total, 62.4%, 40.4% and 12.8% parent(s) at 2, 6 and 12 months, respectively, gave their infant whey-based infant formula. No parental or infant characteristic was found to consistently influence the use of whey-based infant formula. The most common reason reported by parent(s) for changing their infant’s formula to a non whey-based formula was that they perceived their baby as being hungry. Conclusion: The majority of parent(s) commence their infants on whey-based formula, but most change to non whey-based formula before 12 months of age. Parental perception of infant satiety and not healthcare advice was the most common reason for changing from a whey-based to a non whey-based infant formula. Additional research is now required to investigate the effect of whey-based and non whey-based infant formula on infant growth