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Medicine

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Published: March 27th, 2013

DOI:

10.3791/50119

1Imaging Program, Lawson Health Research Institute, 2Department of Anatomy and Cell Biology, Western University, 3Department of Medical Biophysics, Western University

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

1. Maintenance and Propagation of C2C12 Myoblasts

  1. Plate C2C12 myoblasts in a 75 cm2 flask and maintain cells in high glucose Dulbecco's Modified Eagle's Serum (HG-DMEM) supplemented with fetal bovine serum (FBS) to a final concentration of 10%. Do not allow cells to become confluent at any time as this will deplete the myoblastic population. Medium should be changed every other day. Note: always warm medium to 37 °C in a water bath prior to use.
  2. When myoblasts become .......

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Upon 50-60% confluency, C2C12 myoblasts were transiently transfected with the above-mentioned fusion reporter gene construct composed of firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk](Figure 1A). Transfection efficiency was calculated via fluorescence microscopy (Figures 1B,C), making use of the mrfp sequence in our reporter construct. Cell survivability was not affected by labeling with the BLI s.......

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In this study, we have described a fast and reliable molecular imaging, reporter gene approach to non-invasively target myoblasts/MPCs following transplantation. While this study demonstrates the short-term localization of transplanted MPCs via bioluminescense imaging (BLI), the manner in which cells are targeted can, in fact, be easily applied to a longitudinal assessment of cell engraftment, through the implantation of cells that stably express the reporter gene. To this end, our group has generated t.......

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The authors would like to thank Sanjiv Gambhir for the gift of the fluc/mrfp/sr39tk reporter gene. This work was funded by The Stem Cell Network (SCN) of Canada, and The Jesse's Journey Foundation.

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Name Company Catalog Number Comments
Name of reagent Company Catalogue number Comments (optional)
C2C12 myoblasts ATCC
Dulbecco's Modified Eagle's Medium Life Technologies 12800-017
fetal bovine serum Life Technologies 12483020
0.25% Trypsin-EDTA Life Technologies 25200-72
Hanks Balanced Salt Solution Sigma Aldrich H6648
Lipofectamine 2000 Life Technologies 11668-019
Nikon Eclipse TE2000-5 Nikon Instruments Inc.
Xenolight D-luciferin PerkinElmer 122796 40 mg/ml in PBS

  1. Emery, A. E. The muscular dystrophies. Lancet. 359, 687-695 (2002).
  2. Blake, D. J., et al. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol. Rev. 82, 291-329 (2002).
  3. Goldring, K., Partridge, T., Watt, D. Muscle stem cells. J. Pathol. 197, 457-467 (2002).
  4. Partridge, T. A. Cells that participate in regeneration of skeletal muscle. Gene Ther. 9, 752-753 (2002).
  5. Moss, F. P., Leblond, C. P. Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170, 421-435 (1971).
  6. Snow, M. H. An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res. 186, 535-540 (1978).
  7. Kuang, S., Rudnicki, M. A. The emerging biology of satellite cells and their therapeutic potential. Trends Mol. Med. 14 (2), 82 (2008).
  8. Hoffman, E. P., et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N. Engl. J. Med. 318, 1363-1368 (1988).
  9. Ray, P., Tsien, R., Gambhir, S. S. Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects. Cancer Res. 67 (7), 3085 (2007).
  10. Ray, P., et al. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 64, 1323-1330 (2004).
  11. Massoud, T. F., Gambhir, S. S. Molecular imaging in living subjects: seeing fundamental biological processes in a new lights. Genes Dev. 17, 545-580 (2003).
  12. Sicinski, P., et al. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 244, 1578-1580 (1989).
  13. Mattson, M. P. . Pathogenesis of neurodegenerative disorders. In: Contemporary neuroscience. X, 294 (2001).
  14. Andersson, J. E., Bressler, B. H., Ovalle, W. K. Functional regeneration in hind limb skeletal muscle of the mdx mouse. J. Muscle Res. Cell Motil. 9, 499-515 (1988).

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