In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP- labeled Myoblasts  Xiaoyin Xu, Zhong Yang, Qiang Liu, Yaming Wang  Molecular Therapy  Volume 18, Issue 4, Pages 835-842 (April 2010) DOI: 10.1038/mt.2010.3 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Generation of eGFP+ myoblasts. (a) Freshly isolated primary myoblasts labeled with anti-desmin antibody. (b) Myoblasts transduced with eGFP-expressing lentiviral vector and cultured for an additional 10 passages. Bar = 100 µm. eGFP, enhanced green fluorescent protein. Molecular Therapy 2010 18, 835-842DOI: (10.1038/mt.2010.3) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Validation of system fidelity. (a) The relationship of signal intensities with the number of cells in culture. The number of eGFP-labeled myoblasts seeded in each well of 96-well plate in triplicate, from 2,000 to 1,250,000 with a ×5 incremental increase. The insert shows the first three data points in detail. (b) Comparison of the in vivo fluorescence signal of a TA muscle (upper left) and representative sections after tissue harvest (upper right) with fluorescent microscopic images of the same sections (lower two rows). The muscle was harvested at 2-month PCI. (c) In vivo fluorescence images of TA muscles injected with various number of cells. Images were acquired at the time right after cell injection. eGFP, enhanced green fluorescent protein; PCI, post-cell injection; TA, tibialis anterior. Molecular Therapy 2010 18, 835-842DOI: (10.1038/mt.2010.3) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Dynamics of muscle engraftment. (a) An example of engraftment over short term. 4 × 104 eGFP-labeled cells were injected into one location of a TA muscle. In vivo images were acquired from the day 1 till day 14 PCI. The muscle was illuminated using two gooseneck lights with an exposure time of 1,000 ms and binning of 4 × 4. (b) An example of long-term engraftment. The images were acquired from day 1 to day 42 PCI. A total of 5 × 105 eGFP-labeled cells were injected into three positions of a TA muscle. A ring light was used for illumination with an exposure time 1,250 ms and binning of 4 × 4. (c) The change in the amount of fluorescent signal during the course of engraftment of b. Error bar: mean ± SD; n = 6. eGFP, enhanced green fluorescent protein; PCI, post-cell injection; photon counts/sec/M, total photon counts/second/muscle; TA, tibialis anterior. Molecular Therapy 2010 18, 835-842DOI: (10.1038/mt.2010.3) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Evidence of self-renewal and regeneration capability. (a) A representative cycle of cardiotoxin-induced regeneration. 0 day, the image before cardiotoxin injection; 1 day to 21 days, the images of 1–21 days PCTI. (b) The changes in the amount of photon counts over three cycles of cardiotoxin-induced regeneration. Arrows point out the time of cardiotoxin injections. Error bar: mean ± SD; n = 5. PCTI, post-cardiotoxin injection; photon counts/sec/M, total photon counts/second/muscle. Molecular Therapy 2010 18, 835-842DOI: (10.1038/mt.2010.3) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Seven cycles of regeneration induced by cardiotoxin injection, given every 3 weeks. (a) First cycle of cardiotoxin-induced regeneration. D0: image taken before cardiotoxin injection. D1–D14: images taken at 1–14 days PCTI. (b) Seventh cycle of regeneration. (c) Comparison of images before and after regenerations. 5 weeks, the image taken before the first cardiotoxin injection. 8–26 weeks, images taken at the end of each cycle of cardiotoxin-induced regeneration. PCTI, post-cardiotoxin injection. Molecular Therapy 2010 18, 835-842DOI: (10.1038/mt.2010.3) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Histology evidence of myoblast-derived satellite cells. (a,b) eGFP+/Pax7+ donor-derived mononuclear cells located beneath basal lamina in transplanted muscles harvested at (a) 2 months and (b) 1 month PCI double labeled by anti-Pax7 (red) and laminin (purple) antibodies. (c) eGFP+/Ki67+ cells in the regenerating muscle. The muscle was harvested 5 days after cardiotoxin injection and labeled by anti-Ki67 antibody. Bar = 20 µm. eGFP, enhanced green fluorescent protein. Molecular Therapy 2010 18, 835-842DOI: (10.1038/mt.2010.3) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions