Origami: Single-cell 3D shape dynamics oriented along the apico-basal axis of folding epithelia from fluorescence microscopy data

A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets, aided by cell shape changes at the cellular-level. Changes in cell shape must be studied in the context of cell-polarised biomechanical processes within the epithelial sheet. In epithelia with highly curved surfaces, finding single-cell alignment along a biological axis can be difficult to automate in silico. We present ‘Origami’, a MATLAB-based image analysis pipeline to compute direction-variant cell shape features along the epithelial apico-basal axis. Our automated method accurately computed direction vectors denoting the apico-basal axis in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia.

Expansion of Knowledge on OCT1 Variant Activity In Vitro and In Vivo Using Oct1/2−/− Mice

The role of organic cation transporter 1 (OCT1) in humans is gaining attention as data emerges regarding its role in physiology, drug exposure, and drug response. OCT1 variants with decreased in vitro function correlate well with altered exposure of multiple OCT1 substrates in variant carriers. In the current research, we investigate mechanisms behind activity of OCT1 variants in vitro by generating cell lines expressing known OCT1 variants and quantifying membrane OCT1 protein expression with corresponding OCT1 activity and kinetics. Oct knockout mice have provided additional insight into the role of Oct1 in the liver and have reproduced effects of altered OCT1 activity observed in the clinic. To assess the complex effect of Oct1 depletion on pharmacokinetics of prodrug proguanil and its active moiety cycloguanil, both of which are OCT1 substrates, Oct1/2−/− mice were used. Decreased membrane expression of OCT1 was demonstrated for all variant cell lines, although activity was substrate-dependent, as reported previously. Lack of change in activity for OCT1*2 resulted in increased intrinsic activity per pmol of OCT1 protein, particularly for sumatriptan but also for proguanil and cycloguanil. Similar to that reported in humans with decreased OCT1 function, systemic exposure of proguanil was minimally affected in Oct1/2−/− mice. However, proguanil liver partitioning and exposure decreased. Cycloguanil exposure decreased following proguanil administration in Oct1/2−/− mice, as did the systemic metabolite:parent ratio. When administered directly, systemic exposure of cycloguanil decreased slightly; however liver partitioning and exposure were decreased in Oct1/2−/− mice. Unexpectedly, following proguanil administration, the metabolite ratio in the liver changed only minimally, and liver partitioning of cycloguanil was affected in Oct1/2−/− mice to a lesser extent following proguanil administration than direct administration of cycloguanil. In conclusion, these in vitro and in vivo data offer additional complexity in understanding mechanisms of OCT1 variant activity as well as the effects of these variants in vivo. From cell lines, it is apparent that intrinsic activity is not directly related to OCT1 membrane expression. Additionally, in situations with a more complicated role of OCT1 in drug pharmacokinetics there is difficulty translating in vivo impact simply from intrinsic activity from cellular data.

Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated cell competition

The appearance of genetic changes in human pluripotent stem cells (hPSCs) presents a concern for their use in research and regenerative medicine. Variant hPSCs harbouring recurrent culture-acquired aneuploidies display growth advantages over wild-type diploid cells, but the mechanisms yielding a drift from predominantly wild-type to variant cell populations remain poorly understood. Here we show that the dominance of variant clones in mosaic cultures is enhanced through competitive interactions resulting in elimination of wild-type cells. This elimination occurs through corralling and mechanical compression by faster growing variants, causing a redistribution of F-actin and sequestration of YAP in the cytoplasm that induces apoptosis in wild-type cells. Importantly, YAP overexpression in wild-type cells is sufficient to alleviate their loser phenotype. Our results demonstrate that hPSC fate is coupled to mechanical cues imposed by neighbouring cells and reveal that hijacking this mechanism allows variants to achieve clonal dominance in cultures.

Increased Oxidative Stress and Genomic Instability in Peripheral Blood and Bone Marrow of Individuals with Fanconi Anemia,

Abstract 3425 Impaired anti-oxidant defenses and overproduction of reactive oxygen species (ROS) in individuals with Fanconi anemia (FA) are associated with a pro-oxidant state in this population. Cells derived from individuals with FA demonstrate increased sensitivity to DNA damage by ambient oxygen and increased frequency of chromosomal aberrations. Studies in animals and human subjects indicate that higher levels of and increased sensitivity to both ROS and tumor necrosis factor-alpha in individuals with FA play key roles in the pathogenesis of bone marrow failure (BMF) and neoplastic transformation. Accumulation of somatic DNA damage can be assessed using the Glycophorin A assay (GPA), which is designed to detect potentially inactivating mutations caused by chromosome loss, large deletions, or recombination events at the erythrocyte GPA locus. Previous cross-sectional studies in individuals with FA have shown a markedly increased frequency of GPA variant cells, demonstrating increased DNA damage. Knowledge regarding the timecourse of the accumulation of this somatic DNA damage is lacking. The goal of the current study is to evaluate serum ROS levels and accumulation of somatic DNA damage in individuals with FA to better understand the contribution of oxidative stress to disease phenotype and progression to marrow failure or malignant transformation. ROS levels were assessed ex vivo from peripheral blood and bone marrow of individuals with FA and healthy controls. We used a flow cytometric method to quantify ROS level by incubation of samples with CM-H2DCFDA, a cell-permeable fluorescence dye that reacts to a broad spectrum of ROS. Peripheral blood was also serologically typed on first submission using anti-M and anti-N sera. As only heterozygous (MN) individuals are informative, only these individuals were studied further. We used the GPA assay to quantify the accumulation of somatic cell damage in peripheral blood samples as measured by GPA variant cell frequencies. Peripheral blood and/or bone marrow samples from 19 individuals with FA and normal controls were evaluated for ROS on 23 occasions. ROS levels were variable, with 13 of the FA patients demonstrating high levels of ROS as measured by MFI signal strength. Overall ROS levels in individuals with FA were higher than those of normal control individuals (132% of control ROS levels, p<0.09, see Table). When stratified by age, older individuals with FA (age ≥10 years) had significantly higher ROS than normal controls (159% of control levels, p<0.05). Evaluation of 32 children at our center with FA by MN genotyping has identified 13 who are MN heterozygotes eligible for GPA testing. The frequency of NO and NN variant cell frequencies were markedly increased in children with FA (range of 1.2 to 187.5 fold and 0.46 to 201.8 fold over controls respectively). Additionally, longitudinal samples over a 5 year period revealed stable elevation of NO variant frequency with increasing NN variant cell frequency. NN variant frequency was closer to normal in younger individuals with FA and progressively increased with age and development of bone marrow failure.Mean age (years)ROS (% normal control)p-valueAll FA10.21320.09FA age ≥ 10 years12.81590.05FA age < 10 years7.7910.31 We demonstrate that individuals with FA have increased levels of ROS, and that ROS seems to increase with age. This correlates with accumulation of somatic DNA damage over time in this patient population. We hypothesize that ROS levels are low in younger FA individuals and progressively increase with age, resulting in accumulation of DNA damage. Additional longitudinal studies evaluating timecourse, intra-individual variability, and effects of complementation as well as environmental triggers are underway. We expect these studies to identify biomarkers that allow for earlier diagnosis leading to more timely therapy and to elucidate additional targets for novel therapeutic interventions to prevent BMF and myelodysplastic/leukemic transformation. Disclosures: No relevant conflicts of interest to declare.