In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.     

Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies.     

Impaired insulin signaling in an animal model of Niemann-Pick Type C disease

Brain fatty acid-binding protein (FABP7) and PAX6 are both expressed in radial glial cells and have been implicated in neurogenesis and glial cell differentiation. FABP7 and PAX6 have also been postulated to play a role in malignant glioma cell growth and invasion. Here, we address the role of PAX6 in regulating FABP7 gene expression in malignant glioma cells. We report that PAX6 and FABP7 RNA are generally co-expressed in malignant glioma cell lines, tumors and tumor neurospheres. Using the CAT reporter gene assay, we show that FABP7 promoter activity is upregulated by PAX6. Sequential deletion analysis of the FABP7 promoter, combined with gel shift and supershift assays demonstrate the presence of a PAX6 responsive region located upstream of the FABP7 gene, at -862 to -1033 bp. Inclusion of sequences between -1.2 and -1.8 kb reduced CAT activity, suggesting the presence of a repressor element within this region. While PAX6 overexpression did not induce endogenous FABP7 expression in FABP7-negative cells, knock-down of PAX6 in PAX6-positive malignant glioma cells resulted in reduced FABP7 levels. These data provide the first evidence of direct transactivation of the FABP7 proximal promoter by PAX6 and suggest a synergistic mechanism for PAX6 and other co-factor(s) in regulating FABP7 expression in malignant glioma.     

Characterization of the alternate allelic forms of human PAX7

Six different allelic forms of the human neurogenic and myogenic developmental gene, PAX7, have been identified. They are distinguished by the number of tandem tetranucleotide, GAAG, repeats at a polymorphic site within the second intron of the paired box. Within the same intron, a second polymorphic site was found to have variable numbers of a dinucleotide TG repeat. The alleles are identified by a PCR-based method with oligo primers that span the variable regions of the intron. Several of the alleles include a duplicate copy of the entire paired box. Segregation studies demonstrate that the PAX7 alleles are inherited in a Mendelian fashion and that the duplicate copies of the PAX7 paired box region present in some of the alleles are closely linked. This initial study identified differences in the distribution of PAX7 alleles in DNA from patients with the skeletal muscle myopathy, dermatomyositis. Recognition of genetic polymorphism of PAX7 allows new approaches to understanding the role of PAX7 in myogenesis, neurogenesis, and neuromuscular disorders. Received: 13 July 1999 / Accepted: 29 November 1999     

Changes in calpain and caspase gene expression at the mRNA level during bovine muscle satellite cell myogenesis and the correlation between the cell model and the muscle tissue

The calpain proteolytic system plays a central role in cell death and cell signaling. Caspases are a family of proteases implicated in apoptosis. The objective of this study was to explore the regulation and change trend of calpains (CAPN1 and CAPN3) and caspases (caspase-3, caspase-7, and caspase-9) expression at the mRNA level in Luxi cattle skeletal muscle satellite cells during proliferation and differentiation into myotubes. We also sought to assess whether there is a relationship between the muscle satellite cell model and skeletal muscle tissue. Satellite cells were isolated from longissimus dorsi muscle from Luxi cattle and cultured in vitro. Immunofluorescence was used to characterize satellite cells. Our study was divided into three groups: stage one, satellite cells proliferated at 50- and 80-% confluence; stage two, satellite cells differentiated at days 1, 3, 5, 7, and 15; stage three, not the satellite cells but the skeletal muscle tissue. Real-time PCR was used to quantify expression of calpains and the caspases at the mRNA level. These data demonstrated that CAPN1, CAPN3, CASP7, Myf5, and MyoG gene expression significantly increased from satellite cell proliferation to differentiation phases (P < 0.05). In contrast, CASP3 and CASP9 gene expression was significantly down-regulated during myogenesis (P < 0.05). Moreover, we put the CAPN1, CAPN3, CASP3, CASP7, CASP9, Myf5, and MyoG together to say that these genes expression had no significant correlation between the satellite cell model and the skeletal muscle tissue (P > 0.05). Here, we conclude that calpains (CAPN1 and CAPN3), caspases (caspase-3, caspase-7, and caspase-9), and Myf5 and MyoG all have important roles in satellite cell myogenesis. However, there is no relationship between the cell model and muscle tissue.     

An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration

Skeletal muscle tissue provides mechanical force for locomotion of all vertebrate animals. It is prone to damage from acute physical trauma and physiological stress. To cope with this, it possesses a tremendous capacity for rapid and effective repair that is widely held to be accomplished by the satellite cells lying between the muscle fiber plasmalemma and the basement membrane. Cell transplantation and lineage-tracing studies have demonstrated that Pax7-expressing (Pax7(+)) satellite cells can repair damaged muscle tissue repeatedly after several bouts of acute injury. These findings provided evidence that Pax7(+) cells are muscle stem cells. However, stem cells from a variety of other origins are also reported to contribute to myofibers upon engraftment into muscles, questioning whether satellite cells are the only stem cell source for muscle regeneration. Here, we have engineered genetic ablation of Pax7(+) cells to test whether there is any significant contribution to muscle regeneration after acute injury from cells other than this source. We find that such elimination of Pax7(+) cells completely blocks regenerative myogenesis either following injury to the tibialis anterior (TA) muscle or after transplantation of extensor digitorum longus (EDL) muscles into nude mice. As Pax7 is specifically expressed in satellite cells, we conclude that they are essential for acute injury-induced muscle regeneration. It remains to be established whether there is any significant role for stem cells of other origins. The implications of our results for muscle stem cell-based therapy are discussed.     

Asymmetric self-renewal and commitment of satellite stem cells in muscle

Satellite cells play a central role in mediating the growth and regeneration of skeletal muscle. However, whether satellite cells are stem cells, committed progenitors, or dedifferentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre-reporter alleles, we observed that in vivo 10% of sublaminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells gave rise to Pax7(+)/Myf5(+) satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7(+)/Myf5(-) and an apical Pax7(+)/Myf5(+) cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7(+)/Myf5(+) cells exhibited precocious differentiation, Pax7(+)/Myf5(-) cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of neuromuscular diseases.     

Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

Muscle satellite cells

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. The recent discovery of a number of markers expressed by satellite cells has provided evidence that satellite cells, which had long been presumed to be a homogeneous population of muscle stem cells, may not be equivalent. It is possible that a sub-population of satellite cells may be derived from a more primitive stem cell. Satellite cell-derived muscle precursor cells may be used to repair and regenerate damaged or myopathic skeletal muscle, or to act as vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to 3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5) satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells per person. (2) Main functions: repair and maintenance of skeletal muscle. (3) Turnover rate: close to zero in non-traumatic conditions-high in disease or severe trauma.