Musculoskeletal

An immediate application of this technology to musculoskeletal disorders is the monitoring of cell therapies to mitigate the devastating effects of muscular dystrophy. Muscular dystrophy is an inherited disease that is known to result in skeletal muscle weakness and cardiac and respiratory failure, due to chronic bouts of muscle damage and regeneration which eventually exhausts the endogenous pool of stem cells leading to organ failure and death.

Initial transplantation strategies in muscular dystrophy focused primarily on the delivery of myoblasts to the dystrophic muscle. Unfortunately, the therapeutic efficacy of early myoblast transfer studies was limited by massive myoblast cell death observed immediately following in vivo delivery (Cossu and Mavilio, 2000). It has been hypothesized that early cell death after transplantation of myoblasts is due to cellular and humoral immune responses, and the induction of apoptosis (Smythe et al., 2000, 2001). Muscle-derived stem cells (MDSC) have shown a potential ability to repair dystrophic skeletal and cardiac muscle (Deasy, 2002; Jankowski et al., 2002; Torrente et al., 2001). This cell population can undergo in vivo differentiation to regenerate lost myofibers and restore dystrophin expression (Huard et al., 1994; Huard et al., 2003; Ikezawa et al., 2003; Jankowski et al., 2002; Lee et al., 2000; Qu-Petersen et al., 2002). They are also capable of reconstituting the hematopoietic stem cell compartment of lethally irradiated mdx mice (Cao et al., Cao et al.). Both characteristics combined are indicative of a unique stem cell population with a less-committed phenotype than the traditional primary myoblasts used in early transplant studies. Several reports have demonstrated that the MDSC will migrate from the vascu-lature to engraft in dystrophic muscle (Cao et al., 2003; Lee et al., 2000; Torrente et al., 2003, 2001). Embryonic and fetal stem cells have also shown the potential to rescue dystrophic muscle. Intra-arterial injection of wild-type mesoangioblasts (vessel associated fetal stem cells) in a murine model of limb girdle muscle dystrophy resulted in expression of the missing sarcoglycan in more than 50 % of soleus muscle fibers. In addition, vascularly delivered mesoangioblasts have been shown to restore sarcolemmal integrity and results in functional recovery (Galvez et al., 2006; Sampaolesi et al., 2003). This restoration is especially important for the treatment of essential muscles, such as the diaphragm, where impairment results in severe respiratory problems in muscular dystrophy (Chen and Goldhamer, 2003). Overall, a number of studies have found that stem cells have therapeutic potential in skeletal muscle (Brussee et al., 1998; Qu-Petersen et al., 2002; Sampaolesi et al., 2003; Wernig et al., 2000) where transfer efficiencies depend on the cell type, with integrations ranging from 0.1 % to 50 % of a target tissue. In addition, the majority of studies have found that enhancement of chemotatic and mitogenic signals is necessary to increase the efficiency of cell transplantation (Skuk and Tremblay, 2003).

SPIO contrast agents can be utilized to monitor therapeutic muscle stem-cell transplants in a murine model of Duchenne/Becker's muscular dystrophy (mdx mice) (Walter et al., 2004). Early work utilized a dendrimer to shuttle SPIO into MDSC cells, prior to transplantation into mdx mice (Walter et al., 2004). Labeled MDSCs cells were transplanted into the gastrocnemius-plantaris-soleus muscle group of 6-week-old mdx mice. High-resolution MRIs were obtained 24 h, 2, 4, and 11 days post-injection. Distinct regions of signal hypo-intensity were identified in the posterior musculature of animals receiving labeled cell transplants at all time points. Control animals receiving unlabeled cell transplants displayed homogenous images, without the regions of hypo-intensity seen in the experimental animals. Following in vivo imaging, a comparative study was performed with conventional histochemical cell detection techniques. Engrafted cells were detected by analysis of P-galactosidase activity (LacZ), dystrophin expression, and iron content. LacZ expressing fibers were readily identified in regions corresponding to the hypo-intense regions in MR images. Additionally, Prussian blue staining of consecutive serial sections revealed the presence of iron accumulation in many of the LacZ positive fibers, confirming the correlation between the histological location of the cells and MR images. Immunostaining for mini-dystrophin indicated that the engrafted cells restored membrane dystrophin expression and were therefore potentially therapeutic. However, a small number of cells that appeared to be nonmuscle cells also displayed iron-containing granules. Due to the number of macrophages seen in regenerating muscle fibers, it was hypothesized these to be scavenger cells removing the remains of labeled cells that failed to engraft.

Muscle stem and progenitor cells can be efficiently and nontoxically labeled via simple in vitro incubation with SPIO complexes, consisting of commercially available ferumoxides, and the standard molecular biology transfection reagent poly-L-lysine (Cahill et al., 2004a). After 6 h of incubation, 100% cellular labeling was achieved in MDSCs and C2C12 cells, primary myoblasts, and the multipotent human MSCs (Figure 6.7). Similar to magnetodendrimer-labeled cells, ferumoxide-labeled cells could be visualized by high-resolution MRI, following therapeutic transplantation into mdx mice (Cahill et al., 2004a). MRI can also visualize vascular delivery of SPIO MDSC cells (Cahill et al., 2004a). Following arterial delivery of either ferumoxides-labeled myoblasts or MDSCs into dystrophic mice, small, punctuate areas of decreased signal intensity were seen only in the limb musculature of the leg that received labeled cell infusion (Figure 6.8). Histological analyses of the leg musculature showed LacZ expressing muscle-derived stem cells within the vasculature, distributed in patterns corresponding to the MR images. X-gal staining confirmed the presence of stem cell integration in the soleus following vascular delivery (Figure 6.8).

Human Mesenchymal Cells Rat primary myoblast

Prussian Blue Staining For Cell
Figure 6.7. Prussian blue staining of fixed human mesenchymal, rat primary myoblast, muscle-derived stem cells, and C2C12 cells following a 6 h incubation with ferumoxide:poly-L-lysine.

SPIO labeling was found not to be toxic to MDSCs and does not alter the normal growth rate (Cahill et al., 2004a; Walter et al., 2004). The labeled cells differentiated to mature, multinucleated myotubes at rates comparable to unlabeled cells (Cahill et al., 2004b). The resulting myotubes displayed intracellular iron accumulation throughout the length of the myotubes and were otherwise morphologically indistinguishable from unlabeled myotubes. Immunofluorescent analysis of alpha-actinin and desmin expression also revealed that labeled myotubes contain normal sarcomeres (Walter et al., 2004). On transmission electron microscopy images, electron dense areas indicative of iron accumulation could be seen in the endosomal compartments (Cahill et al., 2004a). As previously suggested, trapping of iron oxide inside the endosome reduces the chance of Fenton-like reactions in the myoplasm and the containment of the iron until it can be metabolized (Bulte et al., 2001). In agreement with this hypothesis,

Figure 6.8. Imaging arterial delivery of SPIO-labeled muscle-derived stem cells to a normal mouse hindlimb. Top: 3D MRI of occluded leg (left) and contralateral limb (right) following arterial injection (1 h) of labeled muscle-derived stem cells. Top-right: Transaxial T1 images of the same hindlimbs. Right-middle: Corresponding Prussian blue and XGal of gastrocnemius muscle showing the accumulation of labeled stem cells in major vessels and capillaries of the controlateral limb (x40) in contrast to Fermoxide alone (right-bottom). Modified from Cahill et al. (2004a).

Figure 6.8. Imaging arterial delivery of SPIO-labeled muscle-derived stem cells to a normal mouse hindlimb. Top: 3D MRI of occluded leg (left) and contralateral limb (right) following arterial injection (1 h) of labeled muscle-derived stem cells. Top-right: Transaxial T1 images of the same hindlimbs. Right-middle: Corresponding Prussian blue and XGal of gastrocnemius muscle showing the accumulation of labeled stem cells in major vessels and capillaries of the controlateral limb (x40) in contrast to Fermoxide alone (right-bottom). Modified from Cahill et al. (2004a).

ferumoxide accumulation did not affect cellular viability or alter the normal growth rate of labeled cells in vitro.

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