Growth factors direct mesenchymal stem cell fate and therapeutic potential

Murine mesenchymal stem cells (MSCs) isolated by plastic adherence contain contaminating cells and have poor growth and differentiation. I report a detailed protocol outlining the steps to prospectively isolate a pure and potent MSC population from murine bone marrow based on their expression of stem cell antigen-1 (Sca-1) and platelet derived growth factor- alpha (PDGFRα) (PαS cells) using flow cytometry. PαS MSCs have augmented growth potential and robust tri-lineage differentiation compared to plastic adherent cells. They exert potent immunosuppressive effects on proliferating naive CD4+ T cells, which is mediated via the production of nitric oxide (NO). Nevertheless, prolonged culture results in cellular senescence, loss of adipogenic differentiation and reduced immunosuppressive properties. Addition of growth factors to standard media (SM) produced significant genotypic and phenotypic changes. Cells cultured in SM supplemented with basic fibroblast growth factor (bFGF) and platelet derived growth factor-BB (PDGF-BB) were primed towards fat and cartilage, but had reduced immunosuppressive potential. In contrast, cells cultured with transforming growth factor-beta (TGF-β) had reduced tri-lineage potential but potent immunosuppressive properties that endured despite long term culture. I demonstrate using novel tissue engineering techniques that bFGF PαS MSCs generate substantial 3-D cartilage pellets. These data have implications for MSC therapy in humans

Directional responses following recombinant cytokine stimulation of rainbow trout (Oncorhynchus mykiss) RTS-11 macrophage cells as revealed by transcriptome profiling

Peer reviewedPublisher PD

Starvation alters the liver transcriptome of the innate immune response in Atlantic salmon (Salmo salar)

Peer reviewedPublisher PD

Genetic improvement of feed conversion ratio via indirect selection against lipid deposition in farmed rainbow trout (Oncorhynchus mykiss Walbaum)

The research leading to these results has received funding from the European Union's Seventh Framework Programme (KBBE.2013.1.2-10) under grant agreement n° 613611 FISHBOOST. Moreover, the original data collection was supported by the European Union, Project PROGRESS Q5RS-2001-00994. The staff at Tervo station, Ossi Ritola and Tuija Paananen, are highly acknowledged for fish management. A. Ka., A. Ki., S. M., D. H. and K. R. designed research and wrote the paper; A.Ka analyzed the data and had primary responsibility for the final content. All authors have read and approved the manuscript. The authors declare no conflicts of interest.Peer reviewedPostprintPublisher PD

Recording strategies and selection potential of feed intake measured using the X-ray method in rainbow trout

v2006okBEL/BG

Recording strategies and selection potential of feed intake measured using the X-ray method in rainbow trout

This study examines the way long-term feed intake should be recorded accurately for selective breeding purposes, and estimates selection potential in feed intake using the X-ray method to record individual daily feed intake in rainbow trout (Oncorhynchus mykiss). The analysis showed that the point estimates of daily feed intake displayed low repeatabilities (r = 0.09–0.32). This indicates that a minimum of three repeated records were needed to accurately record average feed intake at a fixed age. To effectively breed for feed intake over the whole growing period, it is necessary to determine average feed intake at different ages, since there were only moderate phenotypic and genetic correlations between average daily feed intake recorded at 140 g, 750 g and 2000 g wet mass. Heritability for average daily feed intake was low (average h2 = 0.10), indicating that modest genetic changes can be obtained in response to selection. It was concluded that selection to genetically change long-term feed intake can be successful, yet repeated observations at several life stages are needed to ensure the accuracy of feed intake estimates and the efficiency of selection

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr–smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)–salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children