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Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification.

ABSTRACT: For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to elucidate how primary human myoblasts and myotubes are modulated by different commonly used serum using FCS (foetal calf serum), (CS-FCS charcoal-stripped FCS, a manufacturing process to remove hormones and growth factors from sera), HS (horse serum) as well as in serum free conditions (DMEM). To characterise the biological impact of the different serum, myoblasts were stimulated with Insulin (100 nM) and Vitamin D (100 nM; 1?,25(OH)2D3, 1?,25-Dihydroxycholecalciferol, Calcitriol), two factors with characterised effects on promoting fusion and protein synthesis or quiescence, respectively in human myoblasts/myotubes. We demonstrate that sera of different origin/formulation differentially affect myoblast proliferation and myotube protein synthesis. Importantly, we showed that quantifying the extent to which Insulin effects myoblasts in vitro is highly dependent upon serum addition and which type is present in the media. Upregulation of mRNA markers for myogenic fusion, Myogenin, with Insulin stimulation, relative to DMEM, appeared dampened at varying degrees with serum addition and effects on p70S6K phosphorylation as a marker of protein synthesis could not be identified unless serum was removed from media. We propose that these asymmetric molecular and biochemical responses in human myoblasts reflect the variable composition of mitogenic and anabolic factors in each of the sera. The results have implications for both the reproducibility and interpretation of results from experimental models in myoblast cells/myotubes.

SUBMITTER: Saini A

PROVIDER: S-EPMC5798781 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification.

Saini Amarjit A Rullman Eric E Lilja Mats M Mandić Mirko M Melin Michael M Olsson Karl K Gustafsson Thomas T

PloS one 20180205 2

For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to eluci ...[more]

PMID: 29401478

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Project description:BackgroundLeft-right asymmetries are a common feature of metazoans and can be found in a number of organs including the nervous system. These asymmetries are particularly pronounced in the simple central nervous system (CNS) of the swimming tadpole larva of the tunicate Ciona, which displays a chordate ground plan. While common pathway elements for specifying the left/right axis are found among chordates, particularly a requirement for Nodal signaling, Ciona differs temporally from its vertebrate cousins by specifying its axis at the neurula stage, rather than at gastrula. Additionally, Ciona and other ascidians require an intact chorionic membrane for proper left-right specification. Whether such differences underlie distinct specification mechanisms between tunicates and vertebrates will require broad understanding of their influence on CNS formation. Here, we explore the consequences of disrupting left-right axis specification on Ciona larval CNS cellular anatomy, gene expression, synaptic connectivity, and behavior.ResultsWe show that left-right asymmetry disruptions caused by removal of the chorion (dechorionation) are highly variable and present throughout the Ciona larval nervous system. While previous studies have documented disruptions to the conspicuously asymmetric sensory systems in the anterior brain vesicle, we document asymmetries in seemingly symmetric structures such as the posterior brain vesicle and motor ganglion. Moreover, defects caused by dechorionation include misplaced or absent neuron classes, loss of asymmetric gene expression, aberrant synaptic projections, and abnormal behaviors. In the motor ganglion, a brain structure that has been equated with the vertebrate hindbrain, we find that despite the apparent left-right symmetric distribution of interneurons and motor neurons, AMPA receptors are expressed exclusively on the left side, which equates with asymmetric swimming behaviors. We also find that within a population of dechorionated larvae, there is a small percentage with apparently normal left-right specification and approximately equal population with inverted (mirror-image) asymmetry. We present a method based on a behavioral assay for isolating these larvae. When these two classes of larvae (normal and inverted) are assessed in a light dimming assay, they display mirror-image behaviors, with normal larvae responding with counterclockwise swims, while inverted larvae respond with clockwise swims.ConclusionsOur findings highlight the importance of left-right specification pathways not only for proper CNS anatomy, but also for correct synaptic connectivity and behavior.

Dataset Information

Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification.

Publications

Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification.

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