SUPPLEMENTARY ONLINE FIGURE LEGENDS
Supplementary Figure 1. Muscle biopsy analysis for glycogen content. A single patient muscle biopsy is divided into approximately ten pieces for analysis (panel A). The pieces are processed by HRLM (6), stained with Richardson’s/ PAS stain, and become ten slides which represent the distribution of glycogen across the entire biopsy and therefore take into account variations in this distribution. Each slide is photographed and analyzed using MetaMorph®. Panel B demonstrates the MetaMorph® analysis of patient A, baseline biopsy. A digital image is captured at a fixed magnification and pixel density. The operator instructs the computer to select all glycogen represented by the purple staining (all selected pixels are highlighted in red, lower left panel). The operator then instructs the computer to select all tissue represented by color (all selected pixels are highlighted in red, lower right panel). The pixel count in the left overlay panel divided by the pixel count in the right overlay panel is 38.9% and represents the percent area of tissue represented by glycogen.
Supplementary Figure 2. Comparison of baseline distributions and shift in the distribution of cell disease stage from baseline to 52 weeks post-treatment. The graph in the upper left-hand corner shows the baseline distribution of cell disease stage for all patients with baseline biopsies. Patients A, C, D, E and F had biopsies at both baseline and week 52 which allowed the evaluation of changes in the myocyte population after enzyme replacement therapy.
Supplementary Figure 3: Satellite cell, capillary density and fiber type analysis.
A. All satellite cells were expressed as a percent of total cell population for all available week 52, post-treatment biopsies (Note: samples required for this analysis were not available for patients B and G). B. The percent of all satellite cells which were activated (myogenin positive) was calculated for all available week 52, post-treatment biopsies. Patient biopsies A and E had the lowest percent activation, which correlated with successful glycogen clearance in these biopsies. C. Frozen sections from post-treatment biopsies were stained with CD31 and capillary counts were performed on representative 200x fields from each biopsy and graphed. There was no significant difference in capillary density between responding and non-responding patient biopsies. D. Frozen sections from post treatment biopsies were stained for type I fibers (ATPase, pH 4.3). Percentage of type I fibers was calculated by MetaMorph®. Patients with the best glycogen clearance (A and E) also had the highest percentage of type I fibers.
SUPPLEMENTARY ONLINE TABLE LEGEND
Supplementary Table 1. Satellite cell activation analysis. All raw data and calculations are listed in the table and graphed in Supplementary Figure 3A and 3B (Note: samples required for this analysis were not available for patients B and G).