isolation-characterization-satellite-cells-from-rat-head

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Radboud university medical center

Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters.

Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein.

In the late stages of muscle development, a unique cell population emerges that is a key player in postnatal muscle growth and muscle regeneration. The location of these cells next to the muscle fibers triggers their designation as satellite cells. During the healing of injured muscle tissue, satellite cells are capable of forming completely new muscle fibers or restoring damaged muscle fibers. A major problem in muscle healing is the formation of dysfunctional scar tissue, which leads to incomplete functional recovery. Therefore, the identification of factors that improve the process of muscle healing and reduce the formation of scar tissue is of great interest. Because satellite cells possess the capability of self-renewal, a unique feature of stem cells, they play a central role in the search for therapies to improve muscle healing. Growth factor-based and (satellite) cell-based therapies are being investigated to treat minor muscle injuries and intrinsic muscle defects. Major muscle injury that involves the loss of muscle tissue requires the use of scaffolds with or without (satellite) cells. Scaffolds are also being developed to generate muscle tissue in vitro. These approaches aim to restore the structure and function of the injured muscle without dysfunctional scarring.

Skeletal muscle development and regeneration.

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

Deficiency of mitochondrial NADH:ubiquinone oxidoreductase (complex I), is associated with a variety of clinical phenotypes such as Leigh syndrome, encephalomyopathy and cardiomyopathy. Circumstantial evidence suggests that increased reactive oxygen species (ROS) levels contribute to the pathogenesis of these disorders. Here we assessed the effect of the water-soluble vitamin E derivative Trolox on ROS levels, and the amount and activity of complex I in fibroblasts of six children with isolated complex I deficiency caused by a mutation in the NDUFS1, NDUFS2, NDUFS7, NDUFS8 or NDUFV1 gene. Patient cells displayed increased ROS levels and a variable decrease in complex I activity and amount. For control cells, the ratio between activity and amount was 1 whereas for the patients this ratio was below 1, indicating a defect in intrinsic catalytic activity of complex I in the latter cells. Trolox treatment dramatically reduced ROS levels in both control and patient cells, which was paralleled by a substantial increase in the amount of complex I. Although the ratio between the increase in activity and amount of complex I was exactly proportional in control cells it varied between 0.1 and 0.8 for the patients. Our findings suggest that the expression of complex I is regulated by ROS. Furthermore, they provide evidence that both the amount and intrinsic activity of complex I are decreased in inherited complex I deficiency. The finding that Trolox treatment increased the amount of complex I might aid the future development of antioxidant treatment strategies for patients. However, such treatment may only be beneficial to patients with a relatively small reduction in intrinsic catalytic defect of the complex.

Mitigation of NADH: ubiquinone oxidoreductase deficiency by chronic Trolox treatment.

The purpose of this study was to establish an in vivo model for muscle regeneration after strain injury in the presence of a fibrotic discontinuity.

Model for muscle regeneration around fibrotic lesions in recurrent strain injuries.

Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF-1 and TGF-beta1 regulate their activity. Donor-derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra-muscular injection. However, a range of other multipotent muscle- and non-muscle-derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases.

Regulatory factors and cell populations involved in skeletal muscle regeneration.

To develop a model for muscle fibrosis based on full-thickness muscle defects, and to evaluate the effects of implanted stromal-derived factor (SDF)-1α-loaded collagen scaffolds.

Skeletal muscle fibrosis: the effect of stromal-derived factor-1&#x03B1;-loaded collagen scaffolds.

The restoration of muscles in the soft palate of patients with cleft lip and/or palate is accompanied by fibrosis, which leads to speech and feeding problems. Treatment strategies that improve muscle regeneration have only been tested in limb muscles. Therefore, in the present study the myogenic potential of muscle progenitor cells (MPCs) isolated from head muscles was compared with that of limb muscles. Muscle progenitor cells were isolated from the head muscles and limb muscles of rats and cultured. The proliferation of MPCs was analysed by DNA quantification. The differentiation capacity was analysed by quantifying the numbers of fused cells, and by measuring the mRNA levels of differentiation markers. Muscle progenitor cells were stained to quantify the expression of paired box protein Pax 7 (Pax-7), myoblast determination protein 1 (MyoD), and myogenin. Proliferation was similar in the head MPCs and the limb MPCs. Differentiating head and limb MPCs showed a comparable number of fused cells and mRNA expression levels of myosin-1 (Myh1), myosin-3 (Myh3), and myosin-4 (Myh4). During proliferation and differentiation, the number of Pax-7(+), MyoD(+), and myogenin(+) cells in head and limb MPCs was equal. It was concluded that head and limb MPCs show similar myogenic capacities in vitro. Therefore, in vivo myogenic differences between those muscles might rely on the local microenvironment. Thus, regenerative strategies for limb muscles might also be used for head muscles.

Myogenic capacity of muscle progenitor cells from head and limb muscles.

Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. These patients are unable to separate the nasal from the oral cavity leading to air loss during speech. Although surgical repair ameliorates soft palate function by joining the clefted muscles of the soft palate, optimal function is often not achieved. The regeneration of muscles in the soft palate after surgery is hampered because of (1) their low intrinsic regenerative capacity, (2) the muscle properties related to clefting, and (3) the development of fibrosis. Adjuvant strategies based on tissue engineering may improve the outcome after surgery by approaching these specific issues. Therefore, this review will discuss myogenesis in the noncleft and cleft palate, the characteristics of soft palate muscles, and the process of muscle regeneration. Finally, novel therapeutic strategies based on tissue engineering to improve soft palate function after surgical repair are presented.

Strategies to improve regeneration of the soft palate muscles after cleft palate repair.

The human NDUFS4 gene encodes an accessory subunit of the first mitochondrial oxidative phosphorylation complex (CI) and, when mutated, is associated with progressive neurological disorders. Here we analyzed primary muscle and skin fibroblasts from NDUFS4(-/-) mice with respect to reactive oxygen species (ROS) levels and mitochondrial morphology. NDUFS4(-/-) fibroblasts displayed an inactive CI subcomplex on native gels but proliferated normally and showed no obvious signs of apoptosis. Oxidation of the ROS sensor hydroethidium was increased and mitochondria were less branched and/or shorter in NDUFS4(-/-) fibroblasts. We discuss the relevance of these findings with respect to previous results and therapy development.

Primary fibroblasts of NDUFS4(-/-) mice display increased ROS levels and aberrant mitochondrial morphology.

Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury.

A rat model for muscle regeneration in the soft palate.

Mitochondria play a key role in signal transduction, redox homeostasis and cell survival, which extends far beyond their classical functioning in ATP production and energy metabolism. In living cells, mitochondrial content ("mitochondrial mass") depends on the cell-controlled balance between mitochondrial biogenesis and degradation. These processes are intricately linked to changes in net mitochondrial morphology and spatiotemporal positioning ("mitochondrial dynamics"), which are governed by mitochondrial fusion, fission and motility. It is becoming increasingly clear that mitochondrial mass and dynamics, as well as its ultrastructure and volume, are mechanistically linked to mitochondrial function and the cell. This means that proper quantification of mitochondrial morphology and content is of prime importance in understanding mitochondrial and cellular physiology in health and disease. This review first presents how cellular mitochondrial content is regulated at the level of mitochondrial biogenesis, degradation and dynamics. Next we discuss how mitochondrial dynamics and content can be analyzed with a special emphasis on quantitative live-cell microscopy strategies.

Regulation and quantification of cellular mitochondrial morphology and content.

Opening of the mitochondrial permeability transition pore (mPTP) is involved in various cellular processes including apoptosis induction. Two distinct states of mPTP opening have been identified allowing the transfer of molecules with a molecular weight <1500 Da or <300 Da. The latter state is considered to be reversible and suggested to play a role in normal cell physiology. Here we present a strategy combining live-cell imaging and computer-assisted image processing allowing spatial visualization and quantitative analysis of reversible mPTP openings ("ΔΨ flickering") in primary mouse myotubes. The latter were stained with the photosensitive cation TMRM, which partitions between the cytosol and mitochondrial matrix as a function of mitochondrial membrane potential (ΔΨ). Controlled illumination of TMRM-stained primary mouse myotubes induced ΔΨ flickering in particular parts of the cell ("flickering domains"). A novel quantitative automated analysis was developed and validated to detect and quantify the frequency, size, and location of individual ΔΨ flickering events in myotubes.

Photo-induction and automated quantification of reversible mitochondrial permeability transition pore opening in primary mouse myotubes.

Reactive oxygen species (ROS) play an important role in both physiology and pathology. Mitochondria are an important source of the primary ROS superoxide. However, accurate detection of mitochondrial superoxide especially in living cells remains a difficult task. Here, we describe a method and the pitfalls to detect superoxide in both mitochondria and the entire cell using dihydroethidium (HEt) and live-cell microscopy.

Live-cell assessment of mitochondrial reactive oxygen species using dihydroethidine.

Rotenone (ROT) is a widely used inhibitor of complex I (CI), the first complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, particularly at high concentrations ROT was also described to display off-target effects. Here we studied how ROT affected in vitro primary murine myotube formation. We demonstrate that myotube formation is specifically inhibited by ROT (10-100nM), but not by piericidin A (PA; 100nM), another CI inhibitor. At 100nM, both ROT and PA fully blocked myoblast oxygen consumption. Knock-down of Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) and, to a lesser extent ROCK1, prevented the ROT-induced inhibition of myotube formation. Moreover, the latter was reversed by inhibiting Raf-1 activity. In contrast, ROT-induced inhibition of myotube formation was not prevented by knock-down of RhoA. Taken together, our results support a model in which ROT reduces primary myotube formation independent of its inhibitory effect on CI-driven mitochondrial ATP production, but via a mechanism primarily involving the Raf-1/ROCK2 pathway.

Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2.

Mitochondrial reactive oxygen species (ROS) production and detoxification are tightly balanced. Shifting this balance enables ROS to activate intracellular signaling and/or induce cellular damage and cell death. Increased mitochondrial ROS production is observed in a number of pathological conditions characterized by mitochondrial dysfunction. One important hallmark of these diseases is enhanced glycolytic activity and low or impaired oxidative phosphorylation. This suggests that ROS is involved in glycolysis (dys)regulation and vice versa. Here we focus on the bidirectional link between ROS and the regulation of glucose metabolism. To this end, we provide a basic introduction into mitochondrial energy metabolism, ROS generation and redox homeostasis. Next, we discuss the interactions between cellular glucose metabolism and ROS. ROS-stimulated cellular glucose uptake can stimulate both ROS production and scavenging. When glucose-stimulated ROS production, leading to further glucose uptake, is not adequately counterbalanced by (glucose-stimulated) ROS scavenging systems, a toxic cycle is triggered, ultimately leading to cell death. Here we inventoried the various cellular regulatory mechanisms and negative feedback loops that prevent this cycle from occurring. It is concluded that more insight in these processes is required to understand why they are (un)able to prevent excessive ROS production during various pathological conditions in humans.

Article	Year
Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein. Cytometry. Part A : the journal of the International Society for Analytical Cytology\| PubMed ID: 17066472	2006
Skeletal muscle development and regeneration. Stem cells and development\| PubMed ID: 17999606	2007
Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 18322173	2008
Mitigation of NADH: ubiquinone oxidoreductase deficiency by chronic Trolox treatment. Biochimica et biophysica acta\| PubMed ID: 18435906	2008
Model for muscle regeneration around fibrotic lesions in recurrent strain injuries. Medicine and science in sports and exercise\| PubMed ID: 19952834	2010
Regulatory factors and cell populations involved in skeletal muscle regeneration. Journal of cellular physiology\| PubMed ID: 20232319	2010
Skeletal muscle fibrosis: the effect of stromal-derived factor-1α-loaded collagen scaffolds. Regenerative medicine\| PubMed ID: 20868329	2010
Myogenic capacity of muscle progenitor cells from head and limb muscles. European journal of oral sciences\| PubMed ID: 22288919	2012
Strategies to improve regeneration of the soft palate muscles after cleft palate repair. Tissue engineering. Part B, Reviews\| PubMed ID: 22697475	2012
Primary fibroblasts of NDUFS4(-/-) mice display increased ROS levels and aberrant mitochondrial morphology. Mitochondrion\| PubMed ID: 23234723	2013
A rat model for muscle regeneration in the soft palate. PloS one\| PubMed ID: 23554995	2013
Regulation and quantification of cellular mitochondrial morphology and content. Current pharmaceutical design\| PubMed ID: 24606803	2014
Photo-induction and automated quantification of reversible mitochondrial permeability transition pore opening in primary mouse myotubes. PloS one\| PubMed ID: 25423172	2014
Live-cell assessment of mitochondrial reactive oxygen species using dihydroethidine. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25631012	2015
Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2. Biochimica et biophysica acta\| PubMed ID: 25827955	2015
Interactions between mitochondrial reactive oxygen species and cellular glucose metabolism. Archives of toxicology\| PubMed ID: 26047665	2015

Sander Grefte

Biography

JoVE Journal Publications

Other Publications