Local mIGF-1 expression enhances the recruitment of bone marrow SP cells to injured muscle. (A) TA muscle of lethally irradiated WT and MLC/mIgf-1 transgenic (Tg) mice reconstituted with c-kit/GFP bone marrow were injected with CTX, and GFP/CD45⁺ cells were analyzed by FACS analysis. Controlateral TA was used as control muscle (Ctr). (B) Increased GFP⁺ cells (yellow arrow) in nonirradiated MLC/mIgf-1 × c-kit/GFP double transgenic mice compared to c-kit/GFP littermates. Necrotic areas are indicated by asterisks. (Inset) GFP⁺ cells near the muscle fibers.

Transgenic mIGF-1 alters local cell populations mobilized by injury. (A) FACS analysis of CD45⁺/Sca-1⁺ cells in bone marrow SP and CD45⁺/Sca-1⁺, CD3⁺/Sca-1⁺ cells in muscle-derived cells from WT and Tg mice before (Ctr) and after (CTX) CTX injection. (B) c-kit⁺/Sca-1⁺ cells in WT and Tg muscle before (Left) and after (Right) CTX injection. Note the appearance of a Sca-1^bright population in regenerating Tg muscle (Lower Right). (C and D) RT-PCR analysis of cell populations isolated by FACS from TA muscles injected with CTX and from controlateral TA (Ctr). (–, RT-PCR buffer without RNA sample; +, positive control; total RNA from embryonic day 9 mouse embryo analyzed for Flk1 and Emx2, and RNA from C2C12 myoblasts analyzed for MyoD). Data are the mean of six separate experiments.

Sca-1 expression defines early muscle cell precursors. (A) Relative desmin expression in primary cultures from injured WT and MLC/mIgf-1 (Tg) muscles. (B) Pax-7, desmin, and MyHC expression in Sca-1⁺ cells isolated by FACS from injured WT and Tg muscle and cultured in GM. (C) Coexpression of Sca-1 and desmin in primary Tg muscle cultures. (Left) Cells harvested after 24 h in GM. (Right) Cells plated at high density and harvested after 4 days in GM. (D) Bone marrow cells isolated from desmin/nLacZ transgenic mice cocultured with primary myoblast monolayers prepared from WT and MLC/mIgf-1 (Tg) mice and stained for β-galactosidase after 4 days. (Right) Representative percentage of blue cells (of a total of 70 cells) in WT and Tg cocultures (mean of six separate experiments).

Regenerating muscle augments local and distal SC compartments. (A) Increased bone marrow SP in WT and MLC/mIgf-1 (Tg) mice after injection with CTX (Right). (B) Fractionation of the bone marrow SP tail from Tg injured muscle. (C) Ablation of the Sca-1⁺/CD45⁺ compartment with 5-FU (mean of four separate experiments).

Transgenic mIGF-1 augments markers of muscle regeneration. (A) FACS analysis of CD11b⁺/Sca-1⁺ cells in bone marrow (Upper) and in muscle-derived cells from injured MLC/mIgf-1 (Tg) mice (Lower and chart). (B) CD11b and β-galactosidase (β-gal) expression in representative primary cells isolated from TA muscle of MLC3f/nLacZ reporter mice. Note muscle-specific β-gal expression only in a subset of small mononucleate cells (Upper). Large cells with flattened morphology never expressed the MLC3f/nLacZ transgene (Lower). (C) Western blot analysis of protein lysates from injured (CTX) and control (Ctr) TA muscles of WT and Tg mice analyzed with Pax-7 antibody. (D) Immunocytochemical analysis of Sca-1 and Pax-7 in transverse sections of TA muscles from WT and Tg mice after CTX injection. Note vessel expressing only Sca-1 (green arrow) and satellite cell expressing Pax7 (red arrow). Nuclei were visualized by Hoechst dye. Data are the mean of six separate experiments.

Model of SC-mediated muscle regeneration enhanced by mIGF-1. Supplemental mIGF-1 enhances the expression of signals from injured muscle (yellow arrows), inducing distal SC mobilization in transplanted bone marrow of irradiated animals and migration to the injured tissue (green–red arrow) or expanding local muscle SC pools. Thereafter, sequential stages of cell commitment to the myogenic lineage (markers in red) are promoted and accelerated by muscle-specific mIGF-1 expression, which induces new markers of regeneration in Sca-1^bright cells. Subsequent loss of SC markers (blue) correlates with activation of myogenic commitment and differentiation programs.