Abstract
Dear Editor,
We would like to respond to the letter by Dr. Yablonka-Reuveni. This letter questioned the method we used to assay acid α-glucosidase (GAA) and therefore the interpretation of our results (Mori et al. 2008). We probed our Western blots with rabbit IgG to human urine GAA, a protein with a molecular mass of 64 kDa (Raben et al. 1998). These Western blots could not determine whether the GAA protein was contributed by donor cells that had become incorporated into the myofibers or donor cells in the interstitium. Similarly, fluorescence-activated cell sorting (FACS) analysis indicated the presence of donor cells within the muscle tissue. Additional experiments, including immunofluorescence analysis and periodic acid-Schiff (PAS) staining, were needed to determine whether the donor cells were incorporated into the myofibers or resided in the interstitium.
In describing our immunofluorescence analysis, we omitted to mention that samples had been fixed with 4% paraformaldehyde in PBS for 30 min, which retains green fluorescent protein (GFP) within cells (Ojima et al. 2004). Although we corrected this mistake in our revised manuscript, this correction was not included in the exPRESS version. Of course, we should study additional cell tracking methods (e.g., male donors to female recipients or lacZ transgenic mice) to clarify that the donor cells were incorporated into myofibers.
Last, we used PAS staining to confirm a reduction in glycogen storage. Because we could not estimate this reduction quantitatively, Dr. Yablonka-Reuveni suggested that cross-sections be stained with PAS, followed by staining for GFP, to show colocalization in the myofibers. Because these experiments may better show the effect of bone marrow transplantation, we intend to stain serial sections in future experiments.
Myeloid cells undergo fusion with myofibers. It is not presently known, however, whether the donor cells induce expression of a missing gene on their incorporation into myofibers. The therapeutic exploitation of the fusion of donor cells with host myofibers remains an option for study. GAA knockout mice are interesting as a new cell tracking method.