Implicating endothelial cell senescence to dysfunction in the ageing and diseased brain

Graves, Sara I.; Baker, Darren J.

doi:10.1111/bcpt.13403

Cited by 73 publications

(64 citation statements)

References 83 publications

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“…1b). Senescent cells can secrete factors including exosomes that can impact surrounding cells as observed with senescent endothelial cells [42], cerebroendothelial cells [43], or fibroblasts [44]. In order to avoid potential artifacts arising from cells that are nearing, or have reached senescence, we suggest that myoblasts under 21 divisions should be used to study the muscle secretome (Fig.…”

Section: Resultsmentioning

confidence: 99%

Optimized method for extraction of exosomes from human primary muscle cells

et al. 2020

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Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 10 6 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation. Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml −1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons. Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.

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Section: Resultsmentioning

confidence: 99%

Optimized method for extraction of exosomes from human primary muscle cells

et al. 2020

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“…1b). Senescent cells can secrete factors including exosomes that can impact surrounding cells as observed with senescent endothelial cells [42], cerobroendothelial cells [43], or broblasts [44]. In order to avoid potential artefacts arising from cells that are nearing, or have reached senescence, we suggest that myoblasts under 21 divisions should be used to study the muscle secretome (Fig.…”

Section: Resultsmentioning

confidence: 99%

Optimized method for extraction of exosomes from human primary muscle cells

Gall

Ouandaogo

Anakor

et al. 2020

Preprint

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Skeletal muscle is increasingly considered as an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can effect physiological changes with impact in different pathological conditions, including regenerative processes, aging and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 x 106 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation.Thus an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml-1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons.Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.

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“…1a, [31]), before they stop dividing and become senescent. Senescent cells can secrete factors including exosomes that can impact surrounding cells as observed with senescent endothelial cells [35], cerobroendothelial cells [36], or fibroblasts [37]. In order to avoid potential artefacts arising from cells that are nearing, or have reached senescence, we suggest that myoblasts under 21…”

Section: Determination Of the Window Of Cell Divisions Suitable To Stmentioning

confidence: 99%

Optimized method for extraction of exosomes from human primary muscle cells

Gall

Ouandaogo

Anakor

et al. 2020

Preprint

View full text Add to dashboard Cite

wordsSkeletal muscle is increasingly considered as an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can effect physiological changes with impact in different pathological conditions, including regenerative processes, aging and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 x 10 6 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation.Thus an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle

show abstract

Implicating endothelial cell senescence to dysfunction in the ageing and diseased brain

Cited by 73 publications

References 83 publications

Optimized method for extraction of exosomes from human primary muscle cells

Optimized method for extraction of exosomes from human primary muscle cells

Optimized method for extraction of exosomes from human primary muscle cells

Optimized method for extraction of exosomes from human primary muscle cells

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