Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation -- Cui et al. 18 (5): 1586 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation
Mol. Biol. Cell Cui et al. 18: 1586

Supplemental Material

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Supplemental Figure 1-1 - Time-course histopathological analysis of implanted cells by vimentin
We performed in vivo time-course histopathological analysis (A-B: 3 h, C-D: 12 h, E-F: 24 h, G-H: 1 week, I-J: 2 weeks, and K-L: 3 weeks) of the EM-E6/E7/hTERT-2 cells without any induction after implantation. The donor cells in the interstitial region at 3 h after implantation are considered to be due to the cell volume used in the injection, and the cells detected at 1 to 3 weeks between myocytes in the muscle bundle (G-L) or muscular fascicle (H, J, and L) as well as in the interstitial tissue can be considered to result from cell migration, as detected by human-specific antibody to vimentin. Scale bars: 500 μm (A, C, E, G, I, K), 100 μm (B, D, F, H, J, L).
Supplemental Figure 1-2
Supplemental Figure 2 - Generation of mdx/mdx scid/scid (mdx-scid) mice and histological characterization of myopathy
Dystrophin-deficient mdx mice (C57BL/10ScSn DMD^mdx/J) were purchased from Jackson ImmunoResearch Laboratories. The mdx-scid mice were produced by mating mdx mice with scid mice (C57BL/6 background, purchased from Charles River, Japan). The genotypes were determined by PCR analysis on DNA obtained from a tail biopsy specimen. Initial denaturation at 94°C for 3 min was followed by 35 cycles of denaturation at 94°C for 3 s, annealing at 57°C for 30 s, and polymerase extension at 72°C for 20 s. A final extension for 10 min at 72°C was followed by storage at 4°C. For each DNA sample to be analyzed, three sets of PCRs were utilized (Supplementary Table 1). One PCR set only allowed amplification of the mdx allele; a second PCR set only allowed amplification of the mdx wild-type allele. The 105-bp DNA products of the PCR reactions were electrophoresed through 4% agarose gels (3 parts Nusieve agarose: 1 part conventional agarose). The third PCR set amplified the scid mice allele. The DNA sample without the mutation should be 64-bp long, and the sample with the mutation should be divided in lengths of 38- and 26-bp after AluI digestion. With each set of PCRs performed, appropriate positive and negative control samples were always analyzed simultaneously, so as to be sure that three sets of PCRs proceeded correctly.
Confirmation for generality of the mutation. (A) The top row shows the PCR amplification produced with mdx-specific primer, the bottom row displays the PCR amplification products generated by the wild-type specific primer. The single bands of 64 bp were amplified by PCR (upper) followed by AluI digestion (lower) in scid mice. Sections of skeletal muscle from control scid mice (B, C) and dystrophic muscles of immunodeficient mice (D, E). C and E are higher magnification of inlets of B and D, respectively. The control scid mice muscle shows close packing of muscle fibers that are nearly uniform in diameter and have peripherally placed nuclei (C, arrowheads). In mdx-scid mice, the muscle fibers show one or more centrally placed nuclei (E, arrowheads), and some areas show excessive atrophy with necrosis of muscle fibers (E, arrow). (B, C, D, E) hematoxylin and eosin stain (HE). Scale bars: 140 μm (B, D), 40 μm (C, E).
Supplemental Figure 3 - Detection of EGFP-positive muscle fibers in mdx-scid mice
EGFP-labeled EM-E6/E7/hTERT-2 cells cultured in absence of any stimuli were directly injected into the thigh muscle of 6- to 8-week-old mdx-scid mice. EGFP-labeled EM-E6/E7/hTERT-2 cells incorporated into newly formed myofibers 3 weeks after implantation.
Supplemental Table