isolation-culture-individual-myofibers-their-satellite-cells-from

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Mini-invasive techniques for gastroesophageal reflux disease (GERD), such as endoscopic injections of inert materials, have been introduced in recent years. However, results are still preliminary. Cell injection has emerged as an alternative strategy in both vesicoureteral reflux and incontinence. Here we report, for the first time, the injection of muscle precursor cells (MPCs) in the gastroesophageal junction (GEJ).

Murine muscle precursor cells survived and integrated in a cryoinjured gastroesophageal junction.

Autologous cell transplantation has been proposed as a possible therapeutic approach for Duchenne dystrophy. In this approach, patients' muscle precursor cells (mpcs) obtained from muscle biopsies would be expanded ex vivo, genetically modified to restore dystrophin expression and then reimplanted in the original donor. Such strategy would have the advantage of bypassing the immune response problem, but on the other hand, it would require a large number of cells because of the poor viability and mobility of transplanted myoblasts. Besides, extensive multiplication of mpcs is difficult and can affect their myogenic ability. Given the key role of inflammation in muscle regeneration, we set out to verify if factors secreted by inflammatory cells could be used to improve in vitro expansion of DMD-mpcs.

Macrophage-secreted factors enhance the in vitro expansion of DMD muscle precursor cells while preserving their myogenic potential.

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.

Selection of multipotent cells and enhanced muscle reconstruction by myogenic macrophage-secreted factors.

The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed onto the hydrogel showed a remarkable sarcomere formation, with the highest percentage (60.0% +/- 3.8) of myotubes exhibiting sarcomeric organization, of myosin heavy chain II and alpha-actinin, after 7 days of culture onto an elastic (15 kPa) hydrogel and a matrigel patterning. In addition, healthy myotubes cultured in these conditions showed a significant membrane-localized dystrophin expression. In this study, the culture substrate has been adapted to human myoblasts differentiation, through an easy and rapid methodology, and has led to the development of in vitro human functional skeletal muscle myotubes useful for clinical purposes and in vitro physiological study, where to carry out a broad range of studies on human muscle physiopathology.

Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes.

Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5â» stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.

Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.

Satellite cells are a heterogeneous population of muscle progenitors with stem cell properties responsible for the regeneration of adult skeletal muscle. Increasing interest in the therapeutic potential of satellite cells has challenged researchers with the need to purify a homogenous population of muscle progenitors. Here we provide a detailed protocol for the isolation of a pure population of satellite cells using fluorescence activated cell sorting. We give specific guidelines to ameliorate the reproducibility of the satellite cell isolation protocol with the goal to standardize procedures across labs. This protocol identifies satellite cells within adult skeletal muscle as an enriched population of Integrin Î±7(+)/CD34(+) double positive cells and CD45, CD31, CD11b, and Sca1 negative (Lin(-)) cells (Integrin Î±7(+)/CD34(+)/Lin(-)). Functional assay shows that Integrin Î±7(+)/CD34(+)/Lin(-) satellite cells possess high myogenic potential and ability to regenerate muscle depleted satellite cells upon transplantation.

Isolation of muscle stem cells by fluorescence activated cell sorting cytometry.

The use of alternative polyadenylation sites is emerging as an important regulator of gene expression. In this issue of Cell Stem Cell, Boutet et al. (2012) report that alternative 3'UTRs of the Pax3 transcript restrict its expression to axial satellite cells through miR-mediated targeting of one of the isoforms.

The long, the short, and the micro: a polyA tale of Pax3 in satellite cells.

Brown adipose tissue (BAT) is an energy-dispensing thermogenic tissue that plays an important role in balancing energy metabolism. Lineage-tracing experiments indicate that brown adipocytes are derived from myogenic progenitors during embryonic development. However, adult skeletal muscle stem cells (satellite cells) have long been considered uniformly determined toward the myogenic lineage. Here, we report that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3'UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake, and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance, and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are downregulated in mice exposed to cold, resulting in de novo generation of satellite cell-derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity.

Article	Year
Murine muscle precursor cells survived and integrated in a cryoinjured gastroesophageal junction. The Journal of surgical research\| PubMed ID: 17583740	2007
Macrophage-secreted factors enhance the in vitro expansion of DMD muscle precursor cells while preserving their myogenic potential. Neurological research\| PubMed ID: 19108751	2010
Selection of multipotent cells and enhanced muscle reconstruction by myogenic macrophage-secreted factors. Experimental cell research\| PubMed ID: 19371636	2009
Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 20473399	2010
Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells. Genes & development\| PubMed ID: 21498568	2011
Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 22130830	2012
The long, the short, and the micro: a polyA tale of Pax3 in satellite cells. Cell stem cell\| PubMed ID: 22385651	2012
MicroRNA-133 controls brown adipose determination in skeletal muscle satellite cells by targeting Prdm16. Cell metabolism\| PubMed ID: 23395168	2013

Alessandra Pasut

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