We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.
Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.
Cell preparation
In Vivo Imaging
At the end of experiments, the mouse is euthanized and the TA muscle is harvested for histology analysis.
We set up a reliable and stable imaging platform for tracking the fluorescence labeled transplanted cells in host skeletal muscle in this study. GFP-labeled myoblasts from GFP transgenic mouse stands for a type of adult stem cells that will be candidates for cell therapy in the future. A constant manipulation including cell number, cell injection, clear background, imaging position and machine parameter is important for gaining high quality image and especially for quantitative analysis.
Fluorescence imaging has many applications in biomedical research as it is non-invasive, easy to use, and has a high throughput. In addition fluorescence imaging can provide a quantitative measurement based on the photon counts, though due to the scattering, attenuation and absorption, light cannot penetrate a large distance in tissue, which is a shortcoming of the technique. Efforts have been underway to use red or near-infrared reporter to increase the penetration depth of light.
Another advantage of fluorescence imaging is that it is translatable to clinics if the specific fluorescent reporter is approved for human use. Compared with MRI, CT, and PET, fluorescence imaging has high flexibility as it does not require expensive hardware and imaging agents. One example is fluorescence-based endoscope and micro-endoscope that have been used in clinics. By combining fluorescence imaging with other modalities, we expect to achieve the capability to acquire both anatomic and functional information about the underlying biological processes.
The work of Z Yang and Y Wang was made possible by grant K02 AR051181 from NIAMS/NIH, grants from Muscular Dystrophy Association and Harvard Stem Cell Institute for Y Wang. The work of Q Zeng and X Xu are supported by a program grant awarded to the Optical Imaging Lab from the Brigham and Women’s Hospital. The authors would like to thank Wen Liu from Department of Anesthesia, BWH, for technical help in cell work.
Material Name | Tip | Company | Catalogue Number | Comment |
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NightOwl LB981 | Berthold Technologies |