Myoblasts isolation

Skeletal myoblasts are viewed as an attractive alternative by some [76]. The first therapeutic trials used skeletal myoblasts obtained under sterile conditions and local anesthesia from 0.5- to 5.0-g muscle biopsy specimens. Individual cells were isolated by digestion with trypsin and collagenase, washed to remove red blood cells and debris, plated, and cultured to obtain the numbers necessary for therapeutic use. 

Ott et al. (29) Rat LAD ligation Autologous skeletal myoblasts vs. BM-MNC SKMB superior to isolated BM-MNC regarding LV function Combination of... 

Looking at the results of both preclinical and clinical studies, we recognize the potential of cardiac cell transplantation to alter outcomes. However, we have to admit that in most cases, we do not understand how different cell types improve LV function. Increases in microvascular density, diastolic and systolic function, and an attenuated remodeling, are all reported after the application of many different cell types, but the exact mechanisms are unclear. Only a few studies address the elec-trophysiologic fate of the injected cells (71,72). In these studies, skeletal myoblasts were found to be isolated from the surrounding myocardium, and they underwent severe... 

Leobon B, Garcin I, Menasche R Vilquin JT Audinat E, Charpak S. Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host. Proc Natl Acad Sci USA 2003 100 7808-781 I. 

Skeletal muscle cells are capable of regeneration and repair more readily than cardiac myocytes owing to the so-called satellite cells that can be readily mobilized at the time of injury (16). They are capable of easy expansion in culture can be harvested easily from the autologous host and are fairly resistant to hypoxia (17). The major drawback is that as the skeletal myocytes mature, they no longer form gap junctions and become electrically isolated, predisposing to re-entry arrhythmias (18). In addition, despite some encouraging data (19), the viability of dissociated myoblasts in suspension injected into myocardial scar deprived of appropriate trophic environment has been called into question (20), In our hands, skeletal myoblasts injected into canine myocardium could not be identified four weeks after implantation, and the needle tract caused fibrosis and scarring (Fig. 2). 

Cummins He did. In the human tissue there seemed to be a striking K sensitivity. However, the same mutation did not seem sensitive to K when expressed in HEK293 cells. But in the myoblasts you have both the wild-type and the mutated allele, plus probably some cardiac Na" channels starting to pop up. One difficulty is that obtaining diseased human tissue is rare. It is going to be even harder to obtain diseased tissue for electrophysiological studies with the neuronal disorders. You can t easily study them in an isolated expression system if there are so many factors needed to precipitate the abnormal activity. 

Immunostaining studies of cells isolated from the contracting areas within the EBs confirmed the presence of cardiac-specific proteins (MHC, sarcomeric a-actinin, des-min, cTnf, ANP). These studies also demonstrated the presence of early-cardiac morphology with a typical early-striated staining pattern. The cells, however, did not exhibit immunoreactivify with anti-nebuhn monoclonal antibodies (mAbs), a specific skeletal muscle sarcomeric protein shown to be expressed early in skeletal myoblast differentiation. 

PG is made in mitochondria and microsomes of animal cells and appears to be primarily converted to DPG. DPG is biosynthesized exclusively on the matrix side of the mitochondrial inner membrane and is found only in this organelle. There is evidence that the rate-limiting step in DPG biosynthesis is the conversion of PA into CDP-DG (G.M. Hatch, 1994). Consistent with this idea, the levels of CTP regulate DPG biosynthesis in cardiac myoblasts (G.M. Hatch, 1996). Using techniques developed by Raetz and co-workers [14], a temperature-sensitive mutant of PG-P synthase in CHO cells was isolated (M. Nishijima 1993). The mutant had only 1% of wild-type PG-P synthase activity at 40°C and exhibited a temperature-sensitive defect in PG and DPG biosynthesis. This mutant was used to show that DPG is required for the NADH-ubiquinone reductase (complex I) activity of the respiratory chain. 

This could not be the case of homofascaplysin A (39) [Fig. (11)], a p-carboline-indole alkaloid isolated from the sponge Hyrtios erecta [56], Indeed, homofascaplysin A (39) showed activity against chloroquine-resistant P. falciparum strains with an IC50 of about 20 ng/mL, but its toxicity toward rat skeletal muscle myoblast cells was estimated to be less than 1 pg/mL, and thus the selectivity index of this molecule is very narrow. 

At nanomolar to micromolar concentrations, yessotoxin has been shown to be toxic to many mammalian cells in culture, including a BE(2)-M17 neuroblastoma cell line [37], a human neuroblastoma cell line [38], HeLa S3 cells [39], rat L6 and mouse BC3H1 skeletal muscle myoblast cell lines [40], P388 mouse leukemia cells [41], 3T3 mouse fibroblasts [42], rat hepatocytes [43], and isolated cerebellar neurons [44]. Yessotoxin did not cause significant mortality in MCE breast cancer cells at nanomolar concentrations, although growth was inhibited [45]. Toxicity to an insect cell line (IPLB-LdFB), derived from a lepidopteran larval fat body, has also been demonstrated [42]. 

The analysis of pADPRS activity during in vitro myogenesis (Fig. 3) demonstrates that a rapid, fourfold increase in activity occurs by day 2. This increase occurs prior to and overlaps the period of myoblast fusion as plotted in Fig. 3 and temporally corresponds to the initiation of the appearance of new muscle-specific RNAs and the initiation of their translation [27]. Coincident with overt and massive expression of these muscle transcripts is a rapid decrease in the pADPRS activity. When fibroblasts are isolated and purified from such cultures and then assayed for pADPRS activity, these muscle-associated fibroblasts exhibit basal levels of pADPRS activity [12]. Thus, the increase in pADPRS activity seems to be associated with the differentiation events of myogenic cells in culture, while the decrease in activity to basal levels is an inherent characteristic of cells which are expressing their differentiated phenotype. 

The isolation of established lines of myoblasts which retain the abUity to fuse and form myotubes has provided a very useful system for studying muscle differentiation [14]. These cells display the same characteristic morphological and biochemical changes as freshly explanted muscle cells in culture and offer the advantage that pure populations of myoblasts and myotubes can be obtained. 

We have examined the involvement of poly(ADP-ribose) in myogenesis using a highly myogenic clone (E63) which had been isolated by Kaufman et al. from the L8 rat skeletal muscle cell line [15]. Usually 7 x 10 myoblasts were plated in 100 mm dishes and grown in DME medium containing 10% horse serum. Under these conditions proliferation of myoblasts slows down on day 4, and cultures become confluent with replication being essentially turned off on day 5. Fusion and synthesis of muscle specific proteins start on day 6-7 and increases until day 10-12 when > 90% of cell nuclei are in multinucleated myotubes. 

Fig. 3. Comparison of poly(ADP-ribosyl)ated proteins isolated from E63 muscle cells and a nonfusing variant (Nf-1). Lane 1) prefusion E63 myoblasts (5 days) 2) differentiated E63 cells (8 days) lane 3) Nf-1 cells maintained for 8 days in culture...

It may be asked whether the changes in the pattern of poly(ADP-ribose) modified proteins are specific for myoblast differentiation. To answer this question we tested a myoblast clone that had lost its capacity to fuse. This clone (Nf-1) was isolated from the E63 myoblast cell line [1 ]. Cultures of Nf-1 were maintained for 8 days and were analyzed for poly(ADP-ribose) modified proteins. Figure 3 shows that the pattern of modified proteins isolated from the nonfusing variant is similar to that of prefusion E63 myoblasts (5 days). The 116 kD modified protein is present in both cultures, whereas the changes in poly(ADP-ribose) acceptor(s) accompanying differentiation are not observed. We have recently observed that the 116 kD modified protein is also formed if differentiation of E63 myoblasts is inhibited by DMSO or UV light. 

The second approach involves the injection of isolated exogenous cells. For example, pluripotent stem cells have been injected into the circulatory system or directly into the heart. Furthermore, embryonic stem cells, skeletal myoblasts, skeletal muscle-derived multimyogenic stem cells, unselected bone marrow stem ceUs, mesenchymal stem cells, endothelial precursor cells, " and adult cardiac stem cells have been shown to differentiate into cardiac phenotypes. Though improved cardiac function has been reported, the underlying mechanism is not clear and the survival rates of the cells tend to be extremely low. This is probably due to the lack of local support for the cells to grow and establish themselves. 

Saxena, A.K., Marler, J., Benvenuto, M., Willital, G.H., Vacanti, J.P., 1999. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers preliminary studies. Tissue Eng. 5, 525—532. 

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