Are cells from a snowman realistic? Cryopreserved tissues as a source for single-cell RNA-sequencing experiments

A recently published study in Genome Biology shows that cells isolated from cryopreserved tissues are a reliable source of genetic material for single-cell RNA-sequencing experiments.Please see related Method article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1171-9

A detailed comparison of mouse and human cardiac development

BACKGROUND: Mouse mutants are used to model human congenital cardiovascular disease. Little is published comparing normal cardiovascular development in mice versus humans. We carried out a systematic comparative analysis of mouse and human fetal cardiovascular development. METHODS: Episcopic fluorescence image capture (EFIC) was performed on 66 wild type mouse embryos from embryonic day (E) 9.5-birth; 2D and 3D datasets were compared with EFIC and magnetic resonance images (MRI) from a study of 52 human fetuses (Carnegie Stage (CS) 13–23). RESULTS: Time course of atrial, ventricular and outflow septation were outlined, and followed a similar sequence in both species. Bilateral vena cavae and prominent atrial appendages were seen in the mouse fetus; in human fetuses, atrial appendages were small, and a single right superior vena cava was present. In contrast to humans with separate pulmonary vein orifices, a pulmonary venous confluence with one orifice enters the left atrium in mice. CONCLUSIONS: The cardiac developmental sequences observed in mouse and human fetuses are comparable, with minor differences in atrial and venous morphology. These comparisons of mouse and human cardiac development strongly support that mouse morphogenesis is a good model for human development

A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations

RATIONALE:Flow cytometry (FCM) of ventricular myocytes (VMs) is an emerging technology in adult cardiac research that is challenged by the wide variety of VM shapes and sizes. Cellular variability and cytometer flow cell size can affect cytometer performance. These two factors of variance limit assay validity and reproducibility across laboratories. Washing and filtering of ventricular cells in suspension are routinely done to prevent cell clumping and minimize data variability without the appropriate standardization. We hypothesize that washing and filtering arbitrarily biases towards sampling smaller VMs than what actually exist in the adult heart. OBJECTIVE:To determine the impact of washing and filtering on adult ventricular cells for cell sizing and FCM. METHODS AND RESULTS:Left ventricular cardiac cells in single-cell suspension were harvested from New Zealand White rabbits and fixed prior to analysis. Each ventricular sample was aliquoted before washing or filtering through a 40, 70, 100 or 200μm mesh. The outcomes of the study are VM volume by Coulter Multisizer and light-scatter signatures by FCM. Data are presented as mean±SD. Myocyte volumes without washing or filtering (NF) served as the "gold standard" within the sample and ranged from 11,017 to 46,926μm3. Filtering each animal sample through a 200μm mesh caused no variation in the post-filtration volume (1.01+0.01 fold vs. NF, n = 4 rabbits, p = 0.999) with an intra-assay coefficient of variation (%CV) of <5% for all 4 samples. Filtering each sample through a 40, 70 or 100μm mesh invariably reduced the post-filtration volume by 41±10%, 9.0±0.8% and 8.8±0.8% respectively (n = 4 rabbits, p<0.0001), and increased the %CV (18% to 1.3%). The high light-scatter signature by FCM, a simple parameter for the identification of ventricular myocytes, was measured after washing and filtering. Washing discarded VMs and filtering cells through a 40 or 100μm mesh reduced larger VM by 46% or 11% respectively (n = 6 from 2 rabbits, p<0.001). CONCLUSION:Washing and filtering VM suspensions through meshes 100μm or less biases myocyte volumes to smaller sizes, excludes larger cells, and increases VM variability. These findings indicate that validity and reproducibility across laboratories can be compromised unless cell preparation is standardized. We propose no wash prior to fixation and a 200μm mesh for filtrations to provide a reproducible standard for VM studies using FCM

IL-11 is a crucial determinant of cardiovascular fibrosis

Fibrosis is a final common pathology in cardiovascular disease1. In the heart, fibrosis causes mechanical and electrical dysfunction1,2 and in the kidney, it predicts the onset of renal failure3. Transforming growth factor β1 (TGFB1) is the principal pro-fibrotic factor4,5 but its inhibition is associated with side effects due to its pleiotropic roles6,7. We hypothesised that downstream effectors of TGFB1 in fibroblasts could be attractive therapeutic targets and lack upstream toxicities. Using integrated imaging-genomics analyses of primary human fibroblasts, we found that Interleukin 11 (IL11) upregulation is the dominant transcriptional response to TGFB1 exposure and required for its profibrotic effect. IL11 and its receptor (IL11RA) are expressed specifically in fibroblasts where they drive non-canonical, ERK-dependent autocrine signalling that is required for fibrogenic protein synthesis. In mice, fibroblast-specific Il11 transgene expression or Il11 injection causes heart and kidney fibrosis and organ failure whereas genetic deletion of Il11ra1 is protective against disease. Thus, inhibition of IL11 prevents fibroblast activation across organs and species in response to a range of important pro-fibrotic stimuli. These data reveal a central role of IL11 in fibrosis and we propose inhibition of IL11 as a new therapeutic strategy to treat fibrotic diseases