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

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.

The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1.

Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily. Many of the genes/proteins which are involved in determining key components of athletic performance have been identified. Naturally occurring mutations in humans as well as gene-transfer experiments in adult animals have shown that altered expression of these genes does indeed affect physical performance. For athletes, however, the gains in performance must be weighed against the health risks associated with the gene-transfer process, whereas the detection of such practices will provide new challenges for the anti-doping authorities.

Gene doping.

Elevated growth hormone (GH) levels lead to increased circulating insulin-like growth factor-I (IGF-I), but the effects on localised muscle IGF-I splice variant expression is not known. The effects of rhGH administration, with or without an acute bout of high resistance exercise, were measured on serum IGF-I and on the mRNA levels of IGF-I splice variants in the vastus lateralis muscle of healthy young men.

Serum IGF-I levels and IGF-I gene splicing in muscle of healthy young males receiving rhGH.

Gene doping is the term given to the potential misuse of gene therapy for the purposes of enhancing athletic performance. Insulin like growth factor-I (IGF-I), the prime target of growth hormone action, is one candidate gene for improving performance. In recent years a number of transgenic and somatic gene transfer studies on animals have shown that upregulation of IGF-I stimulates muscle growth and improves function. This increase in muscle IGF-I is not reflected in measurable increases in circulating IGF-I. Whilst the responses obtained in the animal studies would appear to give clear benefits for performance, the transfer of such techniques to humans still presents many technical challenges. Further challenges will also be faced by the anti doping authorities in detecting the endogenously produced products of enhanced gene expression.

IGF-I and GH: potential use in gene doping.

In an animal model of ALS, intramuscular administration of MGF, the IGF-I Ec gene splice variant, improved muscle strength and increased both motor unit and motor neuron survival. Here we investigated whether there is a deficit in MGF production in the muscles of patients with ALS. We used complementary in vivo and in vitro techniques to study the IGF-I splice variant response of human muscle to exercise or mechanical stretch. We assessed the levels of MGF and IGF-IEa mRNA in muscle biopsy samples from healthy subjects and patients with ALS, before and after exercise. We used primary muscle cells to build three-dimensional collagen constructs and subjected them to a ramp stretch. Patients with ALS had similar baseline levels of MGF and IGF-IEa mRNA to healthy controls. No up-regulation was seen in either group within a short time of a single bout of low intensity exercise. Three-dimensional human muscle constructs also detected no response to a mechanical stretch from either control subjects or ALS. We conclude that the pathology of ALS does not include a deficit in baseline levels of MGF and IGF-IEa mRNA splice variants in muscle.

Investigation of MGF mRNA expression in patients with amyotrophic lateral sclerosis using parallel in vivo and in vitro approaches.

Using a human primary muscle cell culture model the behaviour of myoblasts (satellite cells) cultured in human serum obtained from either young or elderly individuals was studied. Serum was obtained from a total of 13 young (7 males and 6 females aged, 23-36 years) and 9 elderly (4 males and 5 females aged 69-84 years) subjects and used in a number of experiments. Myoblasts were extracted from human muscle biopsy samples taken from the vastus lateralis. In the first experiment myoblasts were isolated immediately after extraction from the biopsy in media containing human sera to examine its effects on the onset and progression of Ki67 and desmin expression. No effect of the age of the serum was observed at 3, 5 or 7 days of culture. In addition, cells were studied that had been expanded initially in optimum myoblast growth medium (GM, containing foetal calf serum and additional growth factors) prior to culture in medium containing 15% human serum. The proportion of proliferating muscle cells coexpressing desmin and Ki67 antigens after 46 h was again similar in the young and old serum conditions. Culturing these myoblasts in media containing 2% human serum to study their fusion and differentiation also resulted in no difference between young and old serum conditions in terms of the percentage of nuclei inside myosin heavy chain positive myotubes. Despite the variability of different samples of myoblasts, the age of the serum donor has no effect on the expression of any measured index.

Sera from young and older humans equally sustain proliferation and differentiation of human myoblasts.

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.

The relationship between age and physiological function remains poorly defined and there are no physiological markers that can be used to reliably predict the age of an individual. This could be due to a variety of confounding genetic and lifestyle factors, and in particular to ill-defined and low levels of physical activity. This study assessed the relationship between age and a diverse range of physiological functions in a cohort of highly active older individuals (cyclists) aged 55-79 years in whom the effects of lifestyle factors would be ameliorated. Significant associations between age and function were observed for many functions. V̇O2 max was most closely associated with age, but even here the variance in age for any given level was high, precluding the clear identification of the age of any individual. The data suggest that the relationship between human ageing and physiological function is highly individualistic and modified by inactivity.

Article	Year
The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1. PLoS biology\| PubMed ID: 15314647	2004
Gene doping. Essays in biochemistry\| PubMed ID: 18384287	2008
Serum IGF-I levels and IGF-I gene splicing in muscle of healthy young males receiving rhGH. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society\| PubMed ID: 18799338	2009
IGF-I and GH: potential use in gene doping. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society\| PubMed ID: 19487147	2009
Investigation of MGF mRNA expression in patients with amyotrophic lateral sclerosis using parallel in vivo and in vitro approaches. Amyotrophic lateral sclerosis : official publication of the World Federation of Neurology Research Group on Motor Neuron Diseases\| PubMed ID: 19572220	2010
Sera from young and older humans equally sustain proliferation and differentiation of human myoblasts. Experimental gerontology\| PubMed ID: 20688143	2010
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
An investigation into the relationship between age and physiological function in highly active older adults. The Journal of physiology\| PubMed ID: 25565071	2015

Cristiana P. Velloso

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