Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days

Introduction: Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. Method: This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Results and discussion: Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 dayspublishedVersio

A linkage map of the Atlantic salmon (Salmo salar) based on EST-derived SNP markers

<p>Abstract</p> <p>Background</p> <p>The Atlantic salmon is a species of commercial and ecological significance. Like other salmonids, the species displays residual tetrasomy and a large difference in recombination rate between sexes. Linkage maps with full genome coverage, containing both type I and type II markers, are needed for progress in genomics. Furthermore, it is important to estimate levels of linkage disequilibrium (LD) in the species. In this study, we developed several hundred single nucleotide polymorphism (SNP) markers for the Atlantic salmon, and constructed male and female linkage maps containing SNP and microsatellite markers. We also investigated further the distribution of male and female recombination events across the genome, and estimated levels of LD between pairs of markers.</p> <p>Results</p> <p>The male map had 29 linkage groups and was 390 cM long. The female map had 30 linkage groups as was 1983 cM long. In total, the maps contained 138 microsatellite markers and 304 SNPs located within genes, most of which were successfully annotated. The ratio of male to female recombination events was either close to zero or very large, indicating that there is little overlap between regions in which male and female crossovers occur. The female map is likely to have close to full genome coverage, while the majority of male linkage groups probably lack markers in telomeric regions where male recombination events occur. Levels of r²increased with decreasing inter-marker distance in a bimodal fashion; increasing slowly from ~60 cM, and more rapidly more from ~12 cM. Long-ranging LD may be consequence of recent admixture in the population, the population being a 'synthetic' breeding population with contributions from several distinct rivers. Levels of r²dropped to half its maximum value (above baseline) within 15 cM, and were higher than 0.2 above baseline for unlinked markers ('useful LD') at inter-marker distances less than 5 cM.</p> <p>Conclusion</p> <p>The linkage map presented here is an important resource for genetic, comparative, and physical mapping of the Atlantic salmon. The female map is likely to have a map coverage that is not far from complete, whereas the male map length is likely to be significantly shorter than the true map, due to suboptimal marker coverage in the apparently small physical regions where male crossovers occur. 'Useful LD' was found at inter-marker distances less than 5 cM.</p

Production of food-grade microcarriers based on by-products from the food industry to facilitate the expansion of bovine skeletal muscle satellite cells for cultured meat production

publishedVersio

Genomic stability through time despite decades of exploitation in cod on both sides of the Atlantic

The mode and extent of rapid evolution and genomic change in response to human harvesting are key conservation issues. Although experiments and models have shown a high potential for both genetic and phenotypic change in response to fishing, empirical examples of genetic responses in wild populations are rare. Here, we compare whole-genome sequence data of Atlantic cod (Gadus morhua) that were collected before (early 20th century) and after (early 21st century) periods of intensive exploitation and rapid decline in the age of maturation from two geographically distinct populations in Newfoundland, Canada, and the northeast Arctic, Norway. Our temporal, genome-wide analyses of 346,290 loci show no substantial loss of genetic diversity and high effective population sizes. Moreover, we do not find distinct signals of strong selective sweeps anywhere in the genome, although we cannot rule out the possibility of highly polygenic evolution. Our observations suggest that phenotypic change in these populations is not constrained by irreversible loss of genomic variation and thus imply that former traits could be reestablished with demographic recovery.publishedVersio

Ticket to spawn: Combining economic and genetic data to evaluate the effect of climate and demographic structure on spawning distribution in Atlantic cod

Climate warming and harvesting affect the dynamics of species across the globe through a multitude of mechanisms, including distribution changes. In fish, migrations to and distribution on spawning grounds are likely influenced by both climate warming and harvesting. The Northeast Arctic (NEA) cod (Gadus morhua) performs seasonal migrations from its feeding grounds in the Barents Sea to spawning grounds along the Norwegian coast. The distribution of cod between the spawning grounds has historically changed at decadal scales, mainly due to variable use of the northern and southern margins of the spawning area. Based on historical landing records, two major hypotheses have been put forward to explain these changes: climate and harvesting. Climate could affect the distribution through, for example, spatial habitat shifts. Harvesting could affect the distribution through impacting the demographic structure. If demographic structure is important, theory predicts increasing spawner size with migration distance. Here, we evaluate these hypotheses with modern data from a period (2000–2016) of increasing temperature and recovering stock structure. We first analyze economic data from the Norwegian fisheries to investigate geographical differences in size of spawning fish among spawning grounds, as well as interannual differences in mean latitude of spawning in relation to changes in temperature and demographic parameters. Second, we analyze genetically determined fish sampled at the spawning grounds to unambiguously separate between migratory NEA cod and potentially smaller sized coastal cod of local origin. Our results indicate smaller spawners farther away from the feeding grounds, hence not supporting the hypothesis that harvesting is a main driver for the contemporary spawning ground distribution. We find a positive correlation between annual mean spawning latitude and temperature. In conclusion, based on contemporary data, there is more support for climate compared to harvesting in shaping spawning ground distribution in this major fish stock in the North Atlantic Ocean

The Atlantic salmon genome provides insights into rediploidization

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.publishedVersio

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. Methods and Findings: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. Conclusions: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation

Assembly and structural analysis of a covalently closed nano-scale DNA cage

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures