Figure 5.

Engrafted CD56⁺/CD29⁺ Cells Respond to Additional Injury by Proliferating and Generating New Human Fibers

(A) Schematic of reinjury experiments. 5,000 human CD56⁺/CD29⁺ cells were transplanted with bupivacaine. One cohort was sacrificed at 5 weeks; one cohort was reinjured with notexin at 5 weeks and sacrificed at 10 weeks; and one cohort was sacrificed at 10 weeks without reinjury. Lightning bolt indicates 18 Gy irradiation; syringe indicates cell transplantation; and skull and crossbones indicates toxin.

(B) Immunostaining for human SPECTRIN and LAMIN A/C on transverse sections at 5 weeks (left), 10 weeks without reinjury (middle), and 10 weeks with notexin reinjury (right). Human muscle: latissimus dorsi, 54-year-old male. Representative sections are shown; n = 4 mice for each group. All sections were co-stained with DAPI. Scale bar, 100 μm.

(C) Immunostaining for pan DYSTROPHIN (DYSTROPHIN) and human DYSTROPHIN (hDYSTROPHIN) at 5 weeks (top row) and at 10 weeks after reinjury with notexin (bottom row). Human muscle: rectus abdominis, 40-year-old female. Representative sections are shown; n = 3 mice for each group. Sections were co-stained with DAPI. Scale bar, 100 μm.

(D) Dot plot showing the number of human SPECTRIN fibers (triangles) identified in individual recipient mice. Results are presented from separate experiments using two different donor muscles: black triangles indicate latissimus dorsi, 54-year-old male; gray triangles indicate rectus abdominis, 40-year-old female. n = 3–4 mice for each group. In the 10 week reinjury animals, 89.8% (SD 5.1%) of the SPECTRIN-positive fibers contained a LAMIN A/C positive nucleus within the same section analyzed for SPECTRIN.

(E) Percentage of total myofibers that express human SPECTRIN after transplantation with human CD56⁺/CD29⁺ cells. Each bar represents the percentage per mouse. Donor cells: black bars represent latissimus dorsi, 54-year-old male; gray bars represent rectus abdominis, 40-year-old female, as in (D). n = 3–4 mice for each group. Statistical analysis was determined using paired Student’s t tests; p values are ^∗ < 0.05, ^∗∗∗ < 0.001. See also Figure S4.

(F) Percentage of DYSTROPHIN-expressing fibers that express human DYSTROPHIN after reinjury. See also Figure S5.

(G) Analysis of neuromuscular junctions on human myofibers after reinjury. Neuromuscular junctions identified by SYNAPTOPHYSIN localization with α-BUNGAROTOXIN (α-BTX). Human origin of myofibers is indicated by human DYSTROPHIN. Arrows point to neuromuscular junctions. Sections were co-stained for DAPI. Scale bar, 20 μm.

See also Figures S6 and S7.