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. 2010 Feb 2;18(4):835–842. doi: 10.1038/mt.2010.3

In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts

Xiaoyin Xu 1, Zhong Yang 2, Qiang Liu 2, Yaming Wang 2,3
PMCID: PMC2862520  PMID: 20125125

Abstract

In vivo fluorescence imaging (FLI) enables monitoring fluorescent protein (FP)-labeled cells and proteins in living organisms noninvasively. Here, we examined whether this modality could reach a sufficient sensitivity to allow evaluation of the regeneration process of enhanced green fluorescent protein (eGFP)-labeled muscle precursors (myoblasts). Using a basic FLI station, we were able to detect clear fluorescence signals generated by 40,000 labeled cells injected into a tibialis anterior (TA) muscle of mouse. We observed that the signal declined to ~25% on the 48 hours of cell injection followed by a recovery starting at the second day and reached a peak of ~45% of the original signal by the 7th day, suggesting that the survived population underwent a limited run of proliferation before differentiation. To assess whether transplanted myoblasts could form satellite cells, we injured the transplanted muscles repeatedly with cardiotoxin. We observed a recovery of fluorescence signal following a disappearance of the signal after each cardiotoxin injection. Histology results showed donor-derived cells located underneath basal membrane and expressing Pax7, confirming that the regeneration observed by imaging was indeed mediated by donor-derived satellite cells. Our results show that FLI is a powerful tool that can extend our ability to unveil complicated biological processes such as stem cell-mediated regeneration.

Introduction

Using fluorescent proteins (FPs) to label cells or molecules has revolutionized our way of studying molecular and cellular mechanisms in biomedical research. The living color feature of FPs enables us to capture biological processes in real-time and to distinguish donor-derived cells without further staining. Advancement on in vivo imaging techniques now has reached a sufficient sensitivity to allow the tracking of FP-labeled cells and molecules in living animals. Since its first report,1 in vivo fluorescent imaging (FLI), also called whole-body imaging, has been used to track FP-labeled tumor cells (for review see ref. 2) and infectious organisms3,4 but has attracted little attention for other applications such as stem cell transplantations.

Factors limiting FLI applications are likely associated with the requirement of external light to excite FPs in this system compared to a bioluminescent imaging (BLI) system. Concerning is that the external light may induce strong autofluorescence and make FLI less quantitative than BLI.5,6 However, how much this problem will actually affect results in applications that require higher sensitivity than tracking tumor growth in some specific tissues, such as muscle, has not been assessed experimentally.

Study of cell transplantation into skeletal muscle has a long history and a renewed interest. Transplantation of myogenic precursors into muscles provides a unique way to deliver genes into diseased muscle cells for therapeutic purposes. Traditionally, to evaluate the outcomes of muscle cell transplantation, animals that received cell transplantation are sacrificed at multiple time points for histological analysis. A gold standard to measure the long-term therapeutic potential of transplanted cells is whether a sub-population can become functional satellite cells. To evaluate the functionality of these proposed satellite cells, either donor-derived satellite cells are isolated for a secondary transplantation, or injury-induced regeneration experiments are performed.7,8,9,10,11 However, it is difficult to confirm that the isolated mononucleated cells are indeed bonafide satellite cells and to distinguish the regenerated donor-derived fibers induced by injury with those spared from the injury. In vivo imaging techniques that allow the repeated tracking of transplanted cells in the same animal could solve these dilemmas. Indeed, recent reports demonstrated that BLI achieved real-time kinetics analysis of muscle engraftment behavior of luciferase-labeled muscle stem cells12 and the measurement of change in a satellite cell population during a specific postnatal development period.13

In the present study, we tracked the process of muscle regeneration by enhanced green fluorescent protein (eGFP)-labeled myoblasts using FLI. Myoblast transplantation is the prototype of muscle cell transplantation and has been studied extensively in preclinical and clinical contexts. Although original clinical trials failed to prove effective muscle engraftment in Duchenne muscular dystrophic patients,14,15,16 much improved results were achieved in mice and nonhuman primates.17,18,19,20 Recently, similar results were confirmed in a clinical trial.21,22 Although multiple studies have shown that transplanted mouse as well as human myoblasts were able to form satellite cells under special conditions such as after preirradiation and cryodamage,7,8,10,23,24,25,26,27 whether the myoblast-derived satellite cells possess similar regeneration capacity as endogenous satellite cells is unclear. We show here that transplantation of a small number of eGFP-labeled myoblasts produced a sufficient amount of fluorescence signal to allow the clear visualization of the engrafted muscle bundles and, in addition, to track the fate of the eGFP-labeled cells during repeated cardiotoxin induced-injuries. We visualized the disappearance and reappearance of eGFP-labeled muscle cells for up to seven cardiotoxin injury cycles. Using histology, we also found donor-derived mononucleated cells located underneath the basal lamina and expressing satellite cell marker Pax7. These data provide explicit evidence that transplanted myoblasts are capable of forming functional satellite cells. Additionally, our study also demonstrates that FLI is a simple, sensitive, and reliable modality that can extend our ability to tackle biological questions with a significant reduction in workload and animal consumption.

Results

Generation of eGFP-expressing myoblasts

Primary myoblasts were isolated from the hind leg muscles of adult C57BL10 mice (Jackson Lab, Bar Harbor, ME) using enzyme digestion followed by preplating as described previously.28 Immunocytochemistry showed that ~98% of resulting cells were positive for desmin, a myogenic marker (Figure 1a). The pooled population were expanded for 10 passages and then infected by lentiviral vectors that expressed eGFP under the control of an elongation factor 1α promoter at a multiple of infection of ~5. Fluorescence-activated cell sorting analysis showed that 95% of cells were positive for eGFP. After the expansion of additional 10 passages, the percentage of eGFP+ cells remained unchanged (Figure 1b).

Figure 1.

Figure 1

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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.

Validation of sensitivity and fidelity of the imaging system

We first determined whether the signal intensity corresponded to the number of eGFP-expressing myoblasts in vitro. eGFP-expressing cells were seeded into a 96-well plate in triplicate, starting with 2,000 cells/well and incrementally increasing to 1,250,000 cells/well. The plate was imaged 3-hours after cell seeding to allow cells to settle down to the bottom of the plate. The result showed a linear relationship between the number of cells and signal intensity measured in photons/second (Figure 2a).

Figure 2.

Figure 2

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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.

In FLI, a concern is that the excitation light source may generate autofluorescence from native tissues that can affect the accurate measurement of the signals from the FPs. To compare the amount of signal from eGFP-labeled cells with that from adjacent host muscle tissue quantitatively, we acquired images from slides with mounted sections of transplanted muscles and measured the fluorescence intensities of eGFP+ areas as well as surrounding non-eGFP areas. These results showed that the signal from eGFP+ tissues is about three times greater than that from eGFP− tissues (Figure 2b). Studies have showed that as long as the fluorescence signal is 20% above the background noise, the signal is detectable.29 Thus, in tibialis anterior (TA) muscles that are ~3 mm diameter in the dorsal-ventral dimension, signals from <1-mm eGFP+ tissue can be detected.

We also compared FLI images with those from fluorescence microscope. The result showed that the eGFP+ regions detected by microscope were faithfully matched with the regions with intense fluorescence in images acquired by FLI (Figure 2b), confirming that the imaging system gives reliable results that reflect the actual distribution of eGFP-labeled cells.

We confirmed that in mice with hair, overwhelming background noise from the hair makes it impossible to observe any fluorescence signal in the muscle. However, this problem was solved either by removing the hair using hair removal reagent like Nair or using nude mice. We also found out that placing the animal on a piece of black paper further reduced background noise. To determine the optimal cell number that could generate a sufficient fluorescence signal to be tracked, we injected 3 µl containing 1 × 104–4 × 105 cells into the TA muscles of severe combined immune deficiency (SCID) mice and acquired the fluorescence image 30 minutes post-cell injection (PCI). TA muscles are the most commonly used muscle to assess the efficiency of cell and gene transplantation into muscle. The result showed that a fluorescence signal was detectable in all muscles, even in the muscles injected with 1 × 104 cells. Due to a massive cell death shortly after cell injection (discussed in the next section), injection of 4 × 104 cells was required to produce a sufficient amount of signal to allow the tracking of the fate of injected cells in TA muscles. Using a set of fixed parameters (exposure time and binning), the fluorescence intensities were identical in multiple measurements from the same animal (data not shown) and among animals proportional to the number of injected eGFP-labeled cells (Figure 2c). The image station is equipped with two types of illumination light sources, including a ring light and a pair of gooseneck optical fibers. We found that since the ring light can be set up in a fixed height all the time, the consistency of imaging results is higher using this illumination source than by using gooseneck optical fibers. On the other hand, the gooseneck optical fibers allow more flexibility to focus the illumination on a region of interest (ROI) for the best results, therefore, they normally produce images with better quality.

Extensor digitorum longus (EDL) muscles are also commonly used for assessing the muscle engraftment capability of transplanted cells. Compared to TA muscles, EDL muscle is smaller (1.5 × 2 mm) and located right underneath the skin of the hind legs. We found out that injection of a total of 2 × 104 eGFP-labeled myoblasts in three positions along the long axis of the EDL muscle allowed the detection of strong fluorescence signals that clearly outline the entire muscle (Supplementary Figure S1). Considering the fact that almost ~80% of injected myoblasts die shortly after injection, the amount of signal was actually produced by only 4,000 cells, thus EDL muscle may provide a paradigm to study the engraftment behaviors of rare populations of stem cells that are difficult to isolate in large numbers.

Tracking muscle engraftment by eGFP-labeled myoblasts

Myoblast transplantation has been studied extensively. We reasoned that the well-defined cell transplantation model would be an excellent example to validate the usefulness and effectiveness of FLI in tracking cell transplantation into muscle. We injected 5 × 105 eGFP-labeled myoblasts at three positions along the long axis of six TA muscles of SCID mice and imaged the hind legs periodically. The results showed that at the first day of PCI, cells produced strong fluorescent signals that sharply declined within 24 hours. Starting at day 3 PCI, the signal increased around the injection sites and then quickly expanded longitudinally. Over the next several days, the eGFP+ area reached its maximal length and the intensity peaked. The remaining fluorescence signal remained unchanged for a week and then slowly declined to stable in a level somewhat lower than the peak (Figure 3a,b). Concomitantly, the boundary of eGFP+ area became sharper and more localized (Figure 3a).

Figure 3.

Figure 3

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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.

To analyze the changes quantitatively, we measured the area and the signal intensity of the eGFP+ region of each TA muscle. To exclude the internal background noise from calculation, we also measured the intensity of an adjacent eGFP− region. We then calculated the total amount of signal and noise, and the net amount of signal yielded by eGFP. As shown in Figure 3c and Supplementary Figure S2, the total signal on the first day decreased ~78% by the 3rd day PCI, indicating that the large portion of injected cells died during this period of time, and confirming the results reported previously.30,31,32,33 The total signal then increased about onefold from day 3 to day 7 and was associated with a quick expansion of eGFP+ area longitudinally, suggesting that surviving cells divided approximately once before fusing to form myofibers. The signal attenuated slowly to 60% of its peak by week 6, which was the longest time measured. Because these animals were immunodeficient, the slow decline at the later time is likely caused by the restructuring of regenerated fibers and downregulation of the elongation factor 1α promoter due probably to differentiation rather than immunorejection. Histology showed that the injection of 5 × 105 myoblasts gave rise to an average 340 ± 127 (mean ± SD) eGFP+ myofibers in the transplanted muscle (Supplementary Figure S3). Interestingly, the number of donor-derived myofibers found in these muscles was much greater (approximately fivefold) than that found in nonirradiated and immunosuppressed muscles of mdx mice transplanted with eGFP-labeled myoblasts reported previously.34 It will be interesting to further investigate whether either the dystrophic environment or immunosuppression is not favorable for myogenic precursors to regenerate.

Revealing formation of functional satellite cells by transplanted myoblasts

To investigate whether transplanted myoblasts could form functional satellite cells, we injected 5 × 105 myoblasts into TA muscle of nude mice using the protocol described in the previous section. The initial engraftment was confirmed by FLI. At the 4–5th week PCI, we injected cardiotoxin into the transplanted TA muscles and then imaged the hind legs periodically. Cardiotoxin has been shown to selectively destroy multinuclear myofibers but spares mononuclear cells35,36 such as satellite cells, thus, it has been used extensively to evaluate the regeneration capacity of myogenic precursors. Our results showed that shortly after cardiotoxin injection, eGFP signal substantially reduced and reached the lowest level by the 3rd day post-cardiotoxin injection, confirming that the majority of donor-derived fibers along with host fibers were destroyed. At the 5th day post-cardiotoxin injection, the eGFP signal started to recover and quickly reached the level before cardiotoxin injection 2 days later (Figure 4a,b), indicating that some donor-derived cells became a reserved population that could be activated by injury and participated in the regeneration of myofibers. We then repeated the experiment for two or more cycles at 3-week intervals and observed the similar cycle of changes in the eGFP signal each time (Figure 4b and Supplementary Figure S4). In one animal, we repeated the experiment seven cycles and observed an almost complete recovery after each cycle (Figure 5a–c). More interestingly, at the end of each degenerating/regenerating cycle not only the total intensity recovered to the previous level but also the shape of eGFP+ area was restored (Figure 5c), suggesting that the regeneration by these cells is most likely restrained in the original myofibers. Thus far, our results demonstrated that these transplanted myoblasts were capable of generating a population of myogenic progenitors that could self-renew, respond to injury, and regenerate damaged fibers, with the functional characteristics of muscle satellite cells.

Figure 4.

Figure 4

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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.

Figure 5.

Figure 5

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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.

Satellite cells are defined by their specific location37 and their expression of satellite cell marker proteins, especially Pax7.38 To further prove that these donor-derived regenerative cells also possess anatomic characteristics of endogenous satellite cells, we histologically analyzed muscles harvested at 1 and 2 months PCI and 5–7 days post-cardiotoxin injection. We observed eGFP+ mononucleated cells located beneath the basal lamina of myofibers and some of them expressing Pax7 (Figure 6a,b). In the sections of muscles that received cardiotoxin injection, we also observed eGFP+ cells expressing Ki67, a marker for proliferating cells (Figure 6c). Together, our observations confirm that transplanted myoblasts were able to form cells with the typical characteristics of bonafide satellite cells, although it is not clear whether the regeneration observed by FLI also reflected the regeneration by other types of donor-derived progenitors that stay in interstitium.

Figure 6.

Figure 6

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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.

Discussion

In this study, we present evidence that in vivo fluorescence imaging is a sensitive and reliable modality to monitor the engraftment behaviors of FP-labeled cells in mouse skeletal muscle. Using this modality, we visualized the dynamics of initial muscle engraftment by cultured myoblasts transplanted intramuscularly and damage-induced muscle regeneration. With the improved accuracy and more quantifiable comparison achieved by this modality, we obtained information that otherwise would be difficult to have found through other approaches.

Our results showed a sharp decline in the fluorescence signal within the first day PCI. EGFP is known for its slow degradation in nature (half-life >24 hours, Clontech, Living Colors User Manual); therefore such a drastic decline of fluorescence signal in a short period of time is unlikely to be caused by a physiological level of fluctuation in promoter activity but rather by accelerated protein degradation mechanisms triggered by cell death. A massive cell death shortly after cell injection in myoblast transplantation was evident by multiple groups.30,31,32,33 However, the primary cause(s) of the early cell death remains unclear. The acquired immune response can be largely excluded from being responsible for this event because transplantation studies in SCID mice have been found to be associated with a similar early cell death as in immunocompetent mice39,40,41,42 and also the observation that there was merely lymphocyte infiltration during the period when early death occurred.43 Since SCID mice were used in our study, our result confirmed these previous reports. Our result showed that there was a 78% reduction in the amount of eGFP signal at day 3 PCI compared to the initial signal, which was similar to the results reported previously using other quantification methods such as quantitative reverse transcription-PCR to measure the Y chromosome content derived from male-donor cells30 or the β-gal activity when β-gal-expressing myoblasts transplanted.39

We also observed that after the initial loss, the fluorescence signal gradually recovered and reached a peak that was 45% of the original level and double its lowest level by day 7 PCI, indicating that some cells survived and proliferated during this period of time, even if only at a modest rate. The images acquired daily during this period showed that diffusion of the fluorescence signal along the long axis of the muscles started at day 4 PCI, suggesting that cells started to fuse into myofibers at that point. No further increase in the size and intensity around the injection site and in the total intensity of whole muscle occurred after day 7 PCI, suggesting no net increase in the number of cells after that time point. Myoblasts expanded in culture have shown to be less efficient for muscle engraftment than freshly isolated satellite cells and some highly selected populations of myogenic cells.9,12,44 It will be interesting to compare whether this is because these other cells proliferate more rapidly during this time frame and/or proliferate for a longer time than myoblasts before entering the terminal differentiation stage or becoming quiescent. It will be also interesting to find out whether an environment with regeneration demands such as a dystrophic muscle promotes more vigorous proliferation. We believe that in vivo imaging techniques will be a very useful tool to address these questions.

One of the advantages for cell-based therapeutic approaches versus gene therapy approaches to treat muscle diseases is that transplanted cells may form satellite cells capable of self-renewal and regeneration. It has been reported that myoblasts transplanted intramuscularly have generated stem cell-like populations that reside in the satellite cell location8,10,23,24,25,30 and can repair damaged muscles7,8,10 after multiple injuries. However, these previous experiments were done either in preirradiated mdx muscles in which host muscle cells had limited capability of regeneration due to the depletion of endogenous satellite and other muscle resident myogenic stem cells or in cryodamaged muscles in which the host satellite cells were damaged along with host fibers. Although there was one study showing that myoblasts transplanted into nonirradiated or cryodamaged muscles generated myogenic precursor but whether these cells were satellite cells was not identified.45 It is not clear whether transplanted myoblasts generate satellite cells when the satellite cells of host are not suppressed or host muscles are not damaged and more importantly, whether these donor-derived satellite cells have the same regenerative capacity as endogenous satellite cells. Considering the fact that it is not feasible to irradiate the muscles of patients before cell transplantation or to cryodamage these muscles, these questions are important and have clinical relevance. In the present study, we observed that in nonirradiated muscles, transplanted myoblasts are capable of repairing injured muscles for as many as seven cycles and some of donor-derived mononuclear cells occupied the specific satellite cell location and expressed the satellite cell marker Pax7. Thus, our study provides evidence for the formation of donor-derived satellite cells in terms of functionality and anatomic location. In addition, the recovery of the fluorescence signal to previous levels after each cycle of degeneration/regeneration in those nonirradiated muscles suggests that these donor-derived satellite cells have the capacity to compete with endogenous satellite cells for regeneration. Moreover, since the images by FLI reached a clarity that enabled the detection of the spatial location of donor-derived myofibers and a sharp boundary of eGFP+ regions, we observed that, during each regeneration cycle, not only did the fluorescence signal recover, but also the location, and size and shape of eGFP+ regions was also roughly restored to the preinjury status, indicating that these donor-derived cells do not migrate and form myofibers elsewhere during the regeneration, a property that also mirrors the regeneration by endogenous satellite cells. Thus, the advantages provided by the experimental modality that we used allow us to draw conclusions for questions that are difficult to be addressed by those methods used in previous studies.7,8,10,30

Compared to other imaging strategies, optical imaging techniques including BLI and FLI do not require expensive equipment or use radioactive probes and, therefore, they are more practical for applications in small animal experiments. BLI and FLI have their own strengths and limitations. BLI captures native light produced inside of organisms, thus, it is virtually background-free. Therefore, it appears to be more sensitive. The major drawback for BLI is the rather poor spatial resolution in the image acquired. It is true that BLI can be used to measure time course, magnitude, and the duration of transplanted cells and transgene products but to unveil a sophisticated biological process, in many cases, a good spatial presentation is highly desirable. Due to much stronger fluorescence signals being emitted by FPs than by oxidized luciferin,2,46 imaging by FLI provides better spatial resolution. An inherent drawback for FLI is the need for an external light to excite FPs. The external lights can induce autofluorescence from native tissues and may affect quantification for signals from nonpermissive deep organs owing to light attenuation when it travels through tissues. However, this problem is reduced when higher wavelength probes such as with excitation spectra in the red and near infrared are used. Due to these concerns, FLIs have not been fully explored for applications such as stem cell transplantation. Muscle is listed as one of the tissues with the highest light transmission.5 Leg muscles, especially TA muscle have been the most popular targets of muscle cell transplantation and gene transfer experiments. We reasoned that some of these projected drawbacks for FLI might not apply to in vivo imaging of leg muscles. To evaluate whether FLI can be used routinely to monitor in vivo behaviors of cell transplantation into muscle, in the present study, we used a basic model of FLI device and experimental conditions that was not set up for the best imaging effect, but was used in real cell transplantation practices. Our results showed that even the basic model of FLI device allows the tracking of the fate of 2 × 104 and 4 × 104 eGFP-labeled myoblasts in EDL and TA muscles, respectively. Considering that 78% of injected cells died initially, the florescence signal was actually emitted by 4,400 or 11,200 cells, respectively. The number of cells was less than that used in most of studies of muscle cell transplantation (3–10 × 105/muscle).7,10,26,27,47 The amount of fluorescence signal from this small number of transplanted cells was sufficient to allow complete analysis and for some of our conclusions, the imaging clarity was critical.

In this study, our goal is to determine whether FLI can be a useful tool to study the engraftment behavior of moyblasts or stem cells after transplantation into skeletal muscle. We successfully demonstrated that a simple FLI allows the monitoring of the fate of a few thousand eGFP-labeled myoblasts transplanted into leg muscles. Like other in vivo imaging techniques, FLI can be used to measure the changes in the quantity of signal over time, which reflects the change in the number of donor-derived cells. We also demonstrated that the projected problems associated with the use of an external light are not a roadblock for the use of FLI, at least in the case of leg muscles. Instead, FLI stands out from BLI in its enhanced clarity of imaging. In addition, our study provided explicit evidence that myoblasts, even when expanded extensively in culture, are still capable of forming satellite cells that possess the functionality, regeneration capacity, and ability to self-renew similar to endogenous satellite cells. Our results also suggested that one of the limiting factors to achieve a high efficiency of muscle engraftment by myoblasts is their limited capability to proliferate extensively before fusing into myofibers. We have proven here that a simple imaging modality can extend our ability to tackle biological questions that are difficult to address by traditional methods and establish a new and simple approach to capture the dynamics of muscle regeneration by transplanted cells. Given the popularity of FP-labeling technique and the simplicity and inexpensive nature of the modality, validation of the feasibility and effectiveness of FLI will have a broad impact in studies of stem cell transplantation.

Materials and Methods

Fluorescence imaging station. We used a planar fluorescence imaging station, NightOwl LB981 (Berthold Technologies, Oak Ridge, TN), to acquire the FLI and its software toolbox, WinLight 3.2, to process the images. The imaging station features a charge-coupled device camera of high sensitivity that can be moved in the vertical direction. The imaging chamber is light-tight to reduce background noise. For the imaging of eGFP, an illumination light source passes through a band-pass filter with a central wavelength of 475 nm to excite the protein, and a band-pass detection filter with a central wavelength of 525 nm selectively captures the emission light of excited eGFP. The excitation and detection filters have a full-width half magnitude of 40 and 10 nm, respectively. Either two gooseneck fibers or a ring shape of light are used to transfer the illumination light onto the subject. Light photograph of subjects can be taken by rotating the detection filter aside.

Isolation of primary myoblasts and stable transduction of eGFP gene. The primary myoblasts were isolated from the leg muscles of adult C57BL10 mice (Jackson Lab) using the protocol by Rando and Blau.28 After the verification of their myogenic identity using immunocytochemistry with anti-desmin antibody, these myoblasts were infected with eGFP-expressing lentiviral vectors. The eGFP-expressing myoblasts' growth with Ham's F10 medium supplemented with 20% of fetal calf serum and 5 ng/ml basic fibroblast growth factor. Before cell transplantation, the myoblasts were first verified for eGFP expression using a fluorescence microscope and then detached with trypsin. After three washes with phosphate-buffered saline and the determination of cell number, the final cell pellet was suspended in phosphate-buffered saline with 1 × 107–5 × 107 cell/ml concentrations.

Cell transplantation and cardiotoxin injection. Animal experiments were carried under the guidance and approval of the Harvard Medical School Standing Committee on animals. Four-to six-week-old male SCID and nude mice were purchased from Taconic (Hudson, NY). After anesthetized by a cocktail of ketamine and xylazine in saline (100 mg/15 mg in 5 ml saline) at a dosage of 100 µl/20 g of body weight, the mouse was placed at prone position. If SCID mice are used, the hair of leg was removed using Nair, a commercially available hair remover (Church & Dwight, Princeton, NJ), and then the leg was wiped with clean water and sterilized. Using a Hamilton syringe, a total of 2–50 × 104 eGFP-labeled myoblasts in 3–10 µl of Hanks were slowly injected into each TA or EDL muscle. The needle was slowly withdrawn in 3 minutes after cell injection. To induce injury, we injected cardiotoxin into the cell transplanted TA muscles starting at 4–5 weeks PCI and repeated the injection for up to seven times with 3-week intervals. Using a Hamilton syringe, 10 ng of cardiotoxin in 30 µl of Hanks was slowly injected into each TA muscle at six equally distributed positions to attempt reaching as many myofibers as possible.

Imaging procedure. The mouse was anesthetized using the protocol described above. For SCID mice, hair of the legs was removed using Nair as described above. To further reduce background noise, the mouse was placed on a piece of nonfluorescent black paper (Strathmore Series 400) in the prone position. To ensure that TA muscles will be imaged at the same position, both legs were positioned 180° apart from each other and 90° relative to body axis. Both feet were taped to the paper with their dorsal sides up. The charge-coupled device camera was set for a field of view of 6.5 cm2 with an estimated specimen height of 0.9 cm. A photographic picture was taken at an exposure time of 10 seconds followed by a fluorescence image at exposure times of 500, 1,000, 1,250, and 2,000 ms, respectively. Images taken at 1,250 ms were subjected to intensity analysis. The mice were imaged periodically for durations up to 8 months.

Quantification of imaging data. We quantified the measurements by both the normalized photon counts and the area of eGFP signals. The ROI containing eGFP signal (ROI) on the fluorescence images was outlined manually, and then the intensity (photon counts/second/mm2) of the ROI as well as the area of the ROI was recorded. To normalize the fluorescent signal from autofluorescence, we also measured the intensity of an adjacent eGFP− region. We then subtracted the photon counts/second/mm2 of ROI by the photon counts/second/mm2 of the eGFP− area and calculated the total photon counts of generated by eGFP+ cells by timing the normalized intensity with the area of eGFP+ region.

Histology. The injected muscles were harvested at various time points PCI and fixed with 4% paraformaldehyde in phosphate-buffered saline for 2 hours in room temperature and then transferred into 30% of sucrose solution overnight before being frozen down with liquid nitrogen. The muscles were cut with cryostat microtome in either cross or longitudinal sections at 12-µm thickness. Digital images of sections were acquired with an Olympus fluorescence microscope (Olympus, Center Valley, PA).

Immunohistochemistry. Muscle sections were incubated with either mouse-anti-Pax7 antibody (1:50 dilution) (Hybridoma Bank, Iowa City, IO) or rabbit-anti-laminin antibody (1:500) (Sigma, St Louis, MO) followed by incubation with the goat-anti-mouse CyTm3-conjugated (Jackson Lab) and goat-anti-rabbit CyTm5-conjugated secondary antibody. Sections of muscles 5–7 days post-cardiotoxin injection were subjected to staining with rabbit-anti-Ki67 antibody (1:500) (BD Pharmingen, Franklin, NJ) followed by incubation with the appropriate CyTm3-conjugated secondary antibodies.

SUPPLEMENTARY MATERIALFigure S1. Engraftment of eGFP-labeled myoblasts in EDL muscles. The images were acquired 1-month post cell injection. Each EDL muscle was injected with 4 ×104 GFP-labeled cells at 3 positions. Two goose-neck lights were used for illumination with an exposure time of 1,250 ms and binning of 4×4.Figure S2. Dynamics of muscle engraftment. Chart shows the results from the six individual muscles collected in Fig. 3c.Figure S3. Histology evidence of muscle engraftment by eGFP-labeled myoblasts. An example of muscle section shows GFP+ donor-derived myofibers. The TA muscle of nude mouse was harvested at 6 weeks post cell injection without immunohistochemistry staining. Scale bar = 100 μm.Figure S4. Evidence of self-renewal and regeneration capability. The chart shows the results of 5 individual muscles collected in Fig. 4b.

Supplementary Material

Figure S1.

Engraftment of eGFP-labeled myoblasts in EDL muscles. The images were acquired 1-month post cell injection. Each EDL muscle was injected with 4 ×104 GFP-labeled cells at 3 positions. Two goose-neck lights were used for illumination with an exposure time of 1,250 ms and binning of 4×4.

Click here for supplemental data (37.3MB, tiff)
Figure S2.

Dynamics of muscle engraftment. Chart shows the results from the six individual muscles collected in Fig. 3c.

Click here for supplemental data (103.5MB, tiff)
Figure S3.

Histology evidence of muscle engraftment by eGFP-labeled myoblasts. An example of muscle section shows GFP+ donor-derived myofibers. The TA muscle of nude mouse was harvested at 6 weeks post cell injection without immunohistochemistry staining. Scale bar = 100 μm.

Click here for supplemental data (37.3MB, tiff)
Figure S4.

Evidence of self-renewal and regeneration capability. The chart shows the results of 5 individual muscles collected in Fig. 4b.

Click here for supplemental data (37.3MB, tiff)

Acknowledgments

We thank Wen Liu (Department of Anesthesia, BWH) for her help in isolation myoblasts and lentiviral vector production and Karen Westerman (Department of Anesthesia, BWH) for her kind gifts of the GFP-expressing lentiviral plasmids and lentiviral genomic plasmids. We thank Paul Allen (Department of Anesthesia, BWH) for his critical reading of this manuscript and scientific advices. We thank Kimberly Lawson (Department of Radiology, BWH) for her English editing. This work was made possible by fundings from NIH (NIAMS 5KO2AR051181), Muscular Dystrophy Association and Harvard Stem Cell Institution to Y.W. and support from the Department of Radiology of BWH to X.X. All authors declared no conflict of interest in the work reported here.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Engraftment of eGFP-labeled myoblasts in EDL muscles. The images were acquired 1-month post cell injection. Each EDL muscle was injected with 4 ×104 GFP-labeled cells at 3 positions. Two goose-neck lights were used for illumination with an exposure time of 1,250 ms and binning of 4×4.

Click here for supplemental data (37.3MB, tiff)
Figure S2.

Dynamics of muscle engraftment. Chart shows the results from the six individual muscles collected in Fig. 3c.

Click here for supplemental data (103.5MB, tiff)
Figure S3.

Histology evidence of muscle engraftment by eGFP-labeled myoblasts. An example of muscle section shows GFP+ donor-derived myofibers. The TA muscle of nude mouse was harvested at 6 weeks post cell injection without immunohistochemistry staining. Scale bar = 100 μm.

Click here for supplemental data (37.3MB, tiff)
Figure S4.

Evidence of self-renewal and regeneration capability. The chart shows the results of 5 individual muscles collected in Fig. 4b.

Click here for supplemental data (37.3MB, tiff)

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