Comparative analysis of discrete exosome fractions obtained by differential centrifugation

Background: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. Methods: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. Results: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. Conclusions: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines

Distribution, structure and function of Nordic eelgrass (<em>Zostera marina</em>) ecosystems:implications for coastal management and conservation

This paper focuses on the marine foundation eelgrass species, Zostera marina, along a gradient from the northern Baltic Sea to the north-east Atlantic. This vast region supports a minimum of 1480 km2 eelgrass (maximum >2100 km2), which corresponds to more than four times the previously quantified area of eelgrass in Western Europe. Eelgrass meadows in the low salinity Baltic Sea support the highest diversity (4–6 spp.) of angiosperms overall, but eelgrass productivity is low (<2 g dw m-2 d-1) and meadows are isolated and genetically impoverished. Higher salinity areas support monospecific meadows, with higher productivity (3–10 g dw m-2 d-1) and greater genetic connectivity. The salinity gradient further imposes functional differences in biodiversity and food webs, in particular a decline in number, but increase in biomass of mesograzers in the Baltic. Significant declines in eelgrass depth limits and areal cover are documented, particularly in regions experiencing high human pressure. The failure of eelgrass to re-establish itself in affected areas, despite nutrient reductions and improved water quality, signals complex recovery trajectories and calls for much greater conservation effort to protect existing meadows. The knowledge base for Nordic eelgrass meadows is broad and sufficient to establish monitoring objectives across nine national borders. Nevertheless, ensuring awareness of their vulnerability remains challenging. Given the areal extent of Nordic eelgrass systems and the ecosystem services they provide, it is crucial to further develop incentives for protecting them

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001.

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Characterization of primary neurospheres generated from mouse ventral rostral hindbrain

Serotonergic (5-HT) neurons of the reticular formation play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders, such as depression and schizophrenia. However, the molecular mechanisms underlying the differentiation of the progenitor cells and the specification of the 5-HT phenotype are not fully understood. A primary neurosphere cell-culture system from mouse ventral rostral hindbrain at embryonic day 12 was therefore established. The generated primary neurospheres comprised progenitor cells and fully differentiated neurons. Bromodeoxyuridine incorporation experiments in combination with immunocytochemistry for neural markers revealed the proliferation capacity of the neural multipotent hindbrain progenitors within neurospheres and their ability to differentiate toward the neuronal lineage and serotonergic phenotype. Gene expression analysis by reverse transcription with the polymerase chain reaction showed that the neurospheres were regionally specified, as reflected by the expression of the transcription factors Gata2 and Pet1. Treatment of dissociated primary neurospheres with exogenous Shh significantly increased the number of 5-HT-immunopositive cells compared with controls, whereas neutralization of endogenous Shh significantly decreased the number of 5-HT neurons. Thus, the primary neurosphere culture system presented here allows the expansion of hindbrain progenitor cells and the experimental control of their differentiation toward the serotonergic phenotype. This culture system is therefore a useful model for in vitro studies dealing with the development of 5-HT neurons

Chromosome 7 and 19 Trisomy in Cultured Human Neural Progenitor Cells

BACKGROUND:Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations. METHODS AND FINDINGS:While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC(+7)) and trisomy 19 (hNPC(+19)), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC(+7) and hNPC(+19) cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (beta(III)-tubulin) in hNPC(+7) and hNPC(+19), using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50-60 population doublings and never showed neoplastic changes. Although hNPC(+7) and hNPC(+19) survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line. CONCLUSIONS:We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications

Human embryonic stem cell-derived neurons establish region-specific, long-range projections in the adult brain

While the availability of pluripotent stem cells has opened new prospects for generating neural donor cells for nervous system repair, their capability to integrate with adult brain tissue in a structurally relevant way is still largely unresolved. We addressed the potential of human embryonic stem cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) to establish axonal projections after transplantation into the adult rodent brain. Transgenic and species-specific markers were used to trace the innervation pattern established by transplants in the hippocampus and motor cortex. In vitro, lt-NES cells formed a complex axonal network within several weeks after the initiation of differentiation and expressed a composition of surface receptors known to be instrumental in axonal growth and pathfinding. In vivo, these donor cells adopted projection patterns closely mimicking endogenous projections in two different regions of the adult rodent brain. Hippocampal grafts placed in the dentate gyrus projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsule into the cervical spinal cord and via the corpus callosum into the contralateral cortex. Interestingly, acquisition of these region-specific projection profiles was not correlated with the adoption of a regional phenotype. Upon reaching their destination, human axons established ultrastructural correlates of synaptic connections with host neurons. Together, these data indicate that neurons derived from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain

Clonal human fetal ventral mesencephalic dopaminergic neuron precursors for cell therapy research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experimentsThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness (formerly Science and Innovation; PLE2009-0101, SAF2010-17167), Comunidad Autónoma Madrid (S2011-BMD-2336), Instituto Salud Carlos III (RETICS TerCel, RD06/0010/0009) and European Union (Excell, NMP4-SL-2008-214706). This work was also supported by an institutional grant from Foundation Ramón Areces to the Center of Molecular Biology Severo Ocho