Donor-derived Hematopoietic Cell Contribution to Myofibers in Acid α-Glucosidase Deficiency: A Promising Progress or Back to the Beginning?

Dear Editor,

The recent issue of the Journal of Histochemistry & Cytochemistry (JHC) reports a new study about hematopoietic contribution to skeletal muscle regeneration in acid α-glucosidase (GAA) knockout mice (Mori et al. 2008). The GAA knockout mouse serves as a model of glycogen storage disease type II (GSDII, also known as Pompe disease). GSDII is one of several glycogen storage disorders caused by abnormalities in the metabolism of glycogen, but GSDII is distinct from other forms in that glycogen accumulates inside the lysosomes. The disease can be categorized into three distinct forms that are determined by the age of onset: the infantile, juvenile, and adult forms. Muscle deterioration is the hallmark of the disorder and, in general, the earlier the onset, the more severe the clinical manifestation of the disease. This study raises hope for future cell-based therapy of GSDII by bone marrow transplantation from healthy donors.

This study takes us back to a controversial issue of skeletal muscle biology that had its peak a few years ago when hematopoietic and bone marrow–derived cells were introduced as magic bullets for cell-based therapy of skeletal muscle. This line of research was based primarily on the identification of host myofibers in which expression of the “enhanced” green fluorescent protein (eGFP) was detected after injection of donor cells expressing eGFP. The donor cells were isolated from mice in which there was strong ubiquitous expression of eGFP driven by a hybrid regulatory construct consisting of the chicken β-actin promoter and cytomegalovirus enhancer (Okabe et al. 1997). Some studies even reported on such donor cells populating the satellite cell niche (and presumably acquiring a myogenic progenitor fate). In some cases, such donor cells were proposed to induce expression of a missing gene on their incorporation into myofibers (Ferrari et al. 1998; Gussoni et al. 1999; LaBarge and Blau 2002; Brazelton et al. 2003; Camargo et al. 2003; Dreyfus et al. 2004). These types of studies have raised tremendous hope among patients suffering from muscle disorders, especially in cases of Duchenne muscular dystrophy, where the lack of dystrophin in the myofiber plasma membrane results in devastating outcomes. Additional studies have cautioned us about the potential of hematopoietic and bone marrow–derived cells to repair skeletal muscles (Lapidos et al. 2004; Sherwood et al. 2004; Wernig et al. 2005). According to the latter studies, even if fusion of such donor cells with host myofibers does occur, the impact on de novo gene expression contributed by the donor cells is questionable.

The new publication in the JHC by Mori and colleagues (2008) again brings this controversial topic to the fore. The authors transplanted bone marrow cells from eGFP mice into GAA knockout mice and interpreted their findings as evidence that the engrafted bone marrow cells contributed to skeletal muscle fiber formation. However, does this study really raise hope that bone marrow–derived cells can do the trick?

The main flaw of the study is the lack of evidence that the donor cells are incorporated into myofibers. It is rather peculiar to show the periphery of myofibers as being GFP⁺ (Figures 2 and 5) but not to show the presence of GFP⁺ myofibers throughout complete cross-sectional areas. It is well recognized that GFP is a soluble product that does not incorporate into the myofiber plasma membrane. It is customary to fix the tissue with an appropriate fixative such as paraformaldehyde before freezing it for cryosectioning, because otherwise, GFP is rapidly lost from the tissue. In some cases, fixation with paraformaldhyde immediately after sectioning may preserve some of the GFP. However, in this study, the tissue was cryosectioned followed by acetone fixation, and GFP is shown only as a fine line at the myofiber periphery, colocalized with the laminin immunolabel of the myofiber basal lamina. The authors offer no explanation as to why they used this approach and why they do not show GFP throughout the myofiber cross-sectional area. Acetone fixation might have created a signal contributed by donor cells that were incorporated into the interstitium between myofibers. At worst, this GFP signal at the myofiber periphery is a technical artifact. The authors must show GFP⁺ myofibers and further enhance the study by using additional means to show incorporation of donor cells into the myofibers. These means can be based for example on monitoring the presence of male-derived nuclei in myofibers of host females by Y-chromosome detection or using donor cells from mice carrying a nuclear lacZ marker. The finding that the CD45⁺:Sca1⁺ population (i.e., hematopoietic cells) isolated from host skeletal muscle also contained donor-derived (GFP⁺) cells (Figure 3) merely indicated engraftment of donor cells within the muscle tissue.

In addition, evidence of enhanced α-glucosidase protein expression in skeletal muscle extracts after injection of donor cells (Figure 1) is not very convincing; the Western blot data depict a very narrow strip cut out from the entire blot and, even within this narrow strip, there is an additional band just above the presumably “specific” one. What are the molecular weights? How does the rest of the blot look? Is the antibody really specific for the protein (after all, it was produced against the human protein)? The authors should have provided additional characterization of the antibody (perhaps such is available but was overlooked). Also, if indeed the band shown is α-glucosidase, is it possible that the protein is contributed by donor cells that have not incorporated into the myofibers, but in fact reside in the interstitium?

Last, the demonstration [by periodic acid-Schiff (PAS) staining] that glycogen levels were reduced in myofibers from GAA knockout mice after bone marrow transplantation (Figure 6) is not free from criticism. The authors do not show equivalent fiber profiles for control and treated animals. The control image (Figure 6A) contains only fibers with large cross-sectional areas, whereas the image of treated muscle (Figure 6B) also contains fibers with small cross-sectional areas (the latter are presumably slow-type fibers), and there is a huge difference in PAS staining between the larger fibers and the small fibers. Without some specific measurements concerning fiber diameter and PAS staining level, it is hard to evaluate the importance, if any, of the data shown in Figure 6. Serial cross-sections stained by PAS and showing GFP⁺ myofibers to depict colocalization of the two items would have been more convincing for claiming a reduction in glycogen storage on bone marrow transplantation.

In an age of information overload where people may have little time to read an entire article closely, the title of this article may lead readers to the conclusion that, indeed, it is well proven that hematopoietic-derived cells can contribute to myofiber repair. However, the publication of this article has instead added more confusion to a long-standing controversial topic about the potential of hematopoietic cells to serve as a cell-based means for skeletal muscle disease management.