isolation-quantitative-immunocytochemical-characterization-primary

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

King's College London

Cambridge Stem Cell Institute

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored.

An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture.

The myogenic behaviour of primary human muscle precursor cells (MPCs) obtained from young (aged 20-25 years) and elderly people (aged 67-82 years) was studied in culture. Cells were compared in terms of proliferation, DNA damage, time course and extent of myogenic marker expression during differentiation, fusion, size of the formed myotubes, secretion of the myogenic regulatory cytokine TGF-β1 and sensitivity to TGF-β1 treatment. No differences were observed between cells obtained from the young and elderly people. The cell populations were expanded in culture until replicative senescence. Cultures that maintained their initial proportion of myogenic cells (desmin positive) with passaging (n = 5) were studied and compared with cells from the same individuals in the non-senescent state. The senescent cells exhibited a greater number of cells with DNA damage (γ-H2AX positive), showed impaired expression of markers of differentiation, fused less well, formed smaller myotubes and secreted more TGF-β. The data strongly suggest that MPCs from young and elderly people have similar myogenic behaviour.

Primary human muscle precursor cells obtained from young and old donors produce similar proliferative, differentiation and senescent profiles in culture.

We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells. The CD56(-) fraction obtained from the first sort was highly enriched in TE-7(+) fibroblasts. Using quantitative analysis of immunofluorescent staining for lipid content, lineage markers and transcription factors, we tested if the purified cell populations could differentiate into adipocytes in response to treatment with either fatty acids or adipocyte-inducing medium. Both treatments caused the fibroblasts to differentiate into adipocytes, as shown by loss of intracellular TE-7, upregulation of the adipogenic transcription factors PPARγ and C/EBPα, and adoption of a lipid-laden adipocyte morphology. By contrast, myogenic cells did not undergo adipogenesis and showed differential regulation of PPARγ and C/EBPα in response to these adipogenic treatments. Our results show that human skeletal muscle fibroblasts are at least bipotent progenitors that can remain as extracellular-matrix-producing cells or differentiate into adipocytes.

Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation.

Survivors of critical illness demonstrate skeletal muscle wasting with associated functional impairment.

Acute skeletal muscle wasting in critical illness.

Unbiased "omics" techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease.

Article	Year
An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society\| PubMed ID: 22511600	2012
Primary human muscle precursor cells obtained from young and old donors produce similar proliferative, differentiation and senescent profiles in culture. Aging cell\| PubMed ID: 23374245	2013
Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation. Journal of cell science\| PubMed ID: 24101731	2013
Acute skeletal muscle wasting in critical illness. JAMA\| PubMed ID: 24108501	2013
Defining the nociceptor transcriptome. Frontiers in molecular neuroscience\| PubMed ID: 25426020	2014

Chibeza C. Agley

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