Name: tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging
Uploaded: 2009-09-21T15:00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

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&#x2019;s Hospital. The authors would like to thank Wen Liu from Department of Anesthesia, BWH, for technical help in cell work.

Cell preparation
<ol>
 <li>Satellite cell isolation 
 <ol style="list-style-type:lower-alpha;">
 <li>Under anesthetic, the limb muscles of GFP transgenic mice (C57BL/ Ka-&beta;actin-EGFP) are removed. After cutting into small pieces, satellite cells are enzymatically dissociated by incubating the minced tissue with 2% dispase collagenase solution for 1 hour at 37&deg;C. </li>
 <li>Neutralize the enzyme digestion with growth media containing Ham F-10 and 20% FBS, dissociate the muscle by repeated triturating with a Pasteur pipette. The cells are filtered through a cell strainer (&oslash;70-100&mu;m) and then plated on a non-coated dish for 1 hour at 37&deg;C.</li>
 <li>Gently remove the supernatant and plate them on a collagen coated dish. Repeat this step 1 hour later to purify the myoblasts. (pre-plate)</li>
 <li>Change the media and plate the cells in time. The final purified myoblasts are cultured in medium containing Ham F-10, 20% FBS, 100 U/ml penicillin, and 100 &mu;g/ml streptomycin (growth medium) at 37&deg;C with 5% CO2, 95% air. </li>
 </ol> 
</li>
<li>Culture and expand the primary myoblasts in growth medium with addition of basic FGF of 10 ng/ml.</li>
<li>For all the in vivo experiments, we inject 5x105 cells per TA muscle. </li>
<li>When harvesting the myoblasts, we detach the cells by using 0.25% trypsin/EDTA and wash them with PBS twice. The cell numbers are counted with a hemocytometer and the cells are resuspended in HBSS at a concentration of 5x104 cells/&mu;l. The concentrated cells in HBSS are placed on ice and injected within 1 hour after collection.</li>
</ol>
In Vivo Imaging
<ol>
<li>Male mdx mice at the age of 4-6 weeks are purchased from Jackson lab and maintained at an animal housing facility.</li>
<li>After about one week of accommodation to the environment, the mice are ready to be implanted with the cultured cells.</li>
<li>Before cell transplantation, the mouse is anesthetized by an intraperitoneal (i.p.) injection of the mixture of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). Then the hair around the TA muscle area is removed by applying Nair to the leg and waiting for 30 seconds to wipe it clean with distilled water.</li>
<li>While the mouse is still under anesthesia, 5x105 cells in 10&mu;l HBSS are slowly injected into each TA muscle at 3 points using a 30 gauge needle.</li>
<li>After step 4, the mouse can be regularly imaged by in vivo fluorescence imaging. The fluorescence imaging station NightOwl LB981 (Berthold Technologies, Inc.), equipped with a high sensitive charge-coupled camera, is used to obtain images from the hinder legs of the mice.</li>
<li>On the day of in vivo imaging, we first check the hair regrowth on the TA muscle. If necessary, we re-apply Nair to remove the hair as described above, after anesthetizing the mouse with the mixture of Ketamine and Xylazine.</li>
<li>While the mouse is under anesthesia, we place the mouse in a prone position on a piece of black non-fluorescent paper (black Artagain paper from Strathmore). To fully expose the TA muscle for better imaging, we tape the hind paws to the paper. </li>
<li>We place the mouse in the imaging chamber and position the hind leg to be imaged at the center of field of view of the camera. We first switch out the emission filter to take a traditional gray-scale photographic picture of the leg with an exposure time of 5 seconds. Readjust the position of the mouse and focus of the camera if necessary. The imaging parameters including exposure time (1,250 ms), specimen height (0.9 cm) etc. are kept constant in the experimental process.</li>
<li>We then switch in the appropriate emission filter for the green fluorescent protein wavelength. We take fluorescence image with an exposure time of 1,250 ms and a binning number of 1. After imaging, the mouse is removed from the imaging chamber to a cage to wait recovery. </li>
<li>For analysis, we draw a region of interest (ROI) over the fluorescent signals to measure their intensity in terms of photons per second. We also measure the size of the ROI in terms of number of pixels as another quantification value. The imaging experiments can be repeated every week or more frequently over a few months, if needed, to acquire longitudinal data. </li>
</ol>
At the end of experiments, the mouse is euthanized and the TA muscle is harvested for histology analysis.

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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.

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<td>NightOwl LB981</td>
<td>&nbsp;</td>
<td>Berthold Technologies</td>
<td>&nbsp;</td>
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tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

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.

Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

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.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

tracking-dynamics-muscle-engraftment-small-animals-vivo-fluorescent

Research

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A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

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In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

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Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

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Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

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In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

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We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
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In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

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Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

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Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...