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First published online 15 June 2004
doi: 10.1242/jcs.01161

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Regeneration of skeletal muscle from transplanted immortalised myoblasts is oligoclonal

¹ Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA
² Clinical and Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK
³ Muscle Cell Biology Group, MRC Clinical Sciences Centre, Imperial College, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK

View larger version (16K): [in a new window]   Fig. 1. Inverse PCR scheme. Integrated retrovirus is shown by a solid line with the 5' and 3' LTR regions represented by boxes at each end and the flanking genomic DNA shown by a dashed line. DNA is digested with SacI restriction enzyme to generate three products, but only those containing the LTR sequence are substrates for primer annealing and PCR amplification. The DNA is ligated under conditions that favour self-ligation and this allows PCR amplification using the forward and reverse primers shown by arrows in opposite directions on the LTR sequence. For each proviral integration two products will be formed, a constant product from amplification of the 3' viral sequence, and a variable product from amplification of genomic DNA flanking the 5' LTR. Retroviral sequence is shown by unbroken lines and flanking genomic DNA by dashed lines.

View larger version (87K): [in a new window]   Fig. 2. IPCR profiles of population A MPCs in culture and after transplantation. (A) IPCR profiles of the cells and muscles following transplantation. Lane C is the cells at the time of implantation and lanes 1-6 and 10-11 are injected TA muscles. Lanes 7-9 are EDL muscles contiguous to injected TA muscles. Yellow asterixes in lanes 2 and 3, and red asterixes in lanes 2, 3 and 11 refer to IPCR products with the same sequence. (B) IPCR profiles of MPCs that emanated from single fibres derived from EDL muscles (lanes 1-4) or TA (lane 5) muscles that had been injected with population A MPCs. (C) IPCR profiles of clones of MPCs derived from population A.

View larger version (53K): [in a new window]   Fig. 3. IPCR profiles of irradiated mdx nu/nu TA muscles injected with 5x105 isolated fibre MPCs (population B). (A) Lane 1 shows retrovirally infected MPCs at passage 5, and lanes 2-4 and 5-8 show profiles from individual muscles injected with cells at passage 5 and 9, respectively. The inner arrow (C) shows the position of the constant-sized product and outer arrows (1-4) indicate products common to the cell and many of the muscle preparations. (B) Right TA muscles of four mice (1-4) were damaged by notexin three weeks after MPC transplantation and muscles were analysed seven days later. L and r refer to the left and right TA muscles; l' and r' refer to the EDL muscle adjacent to the injected TA muscles. (C) Right TA muscles of five mice were damaged with notexin at three and six weeks after cell injection, and all muscles were examined seven days later. L and r refer to the left and right TA muscles; l' and r' refer to the EDL muscle adjacent to the injected TA muscles.

View larger version (59K): [in a new window]   Fig. 4. (A) IPCR profiles of irradiated mdx nu/nu TA muscles of three mice injected with 5x105 isolated fibre MPC (population C). Three weeks after MPC transplantation, the right TA muscles were damaged by notexin injection and muscles were examined seven days later. Profiles are shown of infected cells (population C) at passage four on the day of transplantation (p4), at passage eight (p8) and of muscles of three mice (1l-3r) four weeks after cell transplantation. R refers to the TA of the right leg and l to the TA of the left leg. C refers to the constant band and arrows 1-12 refer to the IPCR products present either in the cell preparations, or in the injected muscles. (B) IPCR profiles of five individual clones (1-5); four ß-gal-positive clones (from population C) and one ß-gal-negative clone (from population B). (C) IPCR profile of the mixed clones before injection (p1), following four weeks in culture (p7) and muscles of three mice that had been transplanted with 5x105 cells total (1x105 cells of each clone). Three weeks after MPC transplantation, right TA muscles were damaged by notexin injection and muscles were analysed seven days later. L and r refer to the left and right TA muscles. Arrows show the constant band (C) and products of 900, 750, 500 and 450 bp (Table 2) that are present within the clones and injected muscles (1l-3r). (D) IPCR profiles of mdx nu/nu TA muscles of three mice injected with 5x105 isolated fibre MPCs (population C). Profiles are shown of infected cells (population C) at passage five on the day of transplantation (p5), at passage 11 (p11) and of muscles of three mice (1l-3r) three weeks after cell transplantation. R refers to the irradiated TA of the right leg and l to the non-irradiated TA of the left leg.

View larger version (116K): [in a new window]   Fig. 5. Transverse cryosections from muscles transplanted with population A MPCs. TA muscles were irradiated and injected with retrovirally infected isolated fibre MPCs (population A). (a) Immunostained for dystrophin and counterstained with hematoxylin. Many dystrophin-positive fibres are present (arrow). Magnificatiox20. (b) Serial section to (a), stained for ß-gal. (c,d) Muscles injected with clone 15 MPCs. (c) Stained for dystrophin and (d) stained for ß-gal. Magnification x10. (e,f) Muscles injected with clone 6 MPCs. (e) Stained for dystrophin and (f) stained for ß-gal. Arrows demonstrate a fibre staining for both dystrophin and ß-gal. Magnification x10.

View larger version (135K): [in a new window]   Fig. 6. Transverse cryosections from muscles transplanted with population C MPCs. TA muscles were irradiated and injected with retrovirally infected isolated fibre MPCs (population C). Three weeks after cell transplantation, the right TA muscles (a,b,e,f) were treated with notexin and the muscles analysed seven days later. (a) Immunostained for dystrophin and counterstained with hematoxylin. Many small, newly regenerated dystrophin-positive fibres are present (small arrow), with a few undamaged dystrophin-negative host fibres (large arrow). The section also contains a large area of undifferentiated cells and necrotic muscle fibres (*). Magnificatiox10. (b) Serial section to (a) stained for ß-gal. (c,d) Left TA muscles [contralateral to (a) and (b)] that had been injected with MPCs but had not been treated with notexin. (c) Stained for dystrophin and (d) stained for ß-gal. Magnification x10. (e) Stained for dystrophin. Magnification x20. (f) Stained for ß-gal. Magnification x20.

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