Name: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia (Video) | JoVE Journal
Uploaded: 2009-01-23T00:00:00.000+00:00
Duration: 9 min 11 s
Description: Learn to deliver embryonic stem cell-derived endothelial cells for treating hindlimb ischemia using bioluminescence imaging. This method enables non-invasive tracking of cell homing and therapeutic effects in vivo.

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5 minutes

The authors thank Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, and the Stanford Small Animal Imaging Core for technical assistance. The authors also thank A.M. Bickford, Inc. for support of veterinary equipment. This research was supported by research grants from the National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), the California Tobacco Related Disease Research Program of the University of California (15IT-0257 and 1514RT-0169 ), and the California Institute for Regenerative Medicine (RS1-00183).
N.H. is supported by a fellowship from the American Heart Association. can Heart Association.

1. Differentiation of murine ESCs into endothelial cells
<ol>
<li>The protocol for differentiating ESCs into endothelial cells is described elsewhere and is not the focus of this protocol2,3. Briefly, the cells are allowed to differentiate, and the cells that are positive for the endothelial markers such as CD31 or vascular endothelial cadherin (VE-cadherin) are then purified by fluorescence activated cell sorting (FACS).</li>
</ol>
2. Construction of the Double Fusion Reporter Gene and Lentiviral Transduction
<ol>
<li>Bioluminescence can be used to track cells that are modified to express reporter genes such as firefly luciferase (fluc). For this application, our cells contain both fluc and enhanced green fluorescence protein (eGFP) fusion gene under the control of an internal ubiquitin promoter. In order to modify the cells, the transgene lentiviral vector containing key optimized genetic elements to fulfill bio-safety criteria as well as increased transduction efficiency, pFU-FG vector developed in our lab carrying the fluc-eGFP fusion reporter gene can be used to stably transduce the cells after differentiation. This fusion construct enables both bioluminescence and fluorescence tracking of the transplanted cells. The procedure for generating virus particles, transduction and production of labeled cells that express reporter genes is described elsewhere4.</li>
</ol>
3. Transplantation of ESC-derived endothelial cells to the ischemic hindlimb
<ol>
<li>Begin the procedure by preparing a mouse that has undergone hindlimb ischemia for cell transplantation. To do this, place the mouse into the anesthesia induction chamber containing 1–3% isoflurane in 100% oxygen at a flow rate of 1L/min. </li>
<li>Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber.</li>
<li>Then, place the animal in the supine position onto the operating table and connect it to a continuous flow of 1–3% isoflurane in 100% oxygen at a flow rate of 1L/min.</li>
<li>Wipe the skin of the hindlimb with three alternating betadine and alcohol scrubs.</li>
<li>Once the skin is cleaned, obtain one million ESC-derived endothelial cells in 30 μL of phosphate buffered saline (PBS). Load these cells into a 28 gauge needle.</li>
<li>When the cells are ready, gently lift and extend the hindlimb to better visualize the location of the gastrocnemius muscle. While the leg is extended, insert the needle through the skin into the underlying muscle. Take care not to approach the bone. Gently and slowly inject the 30 μL cell mixture into the gastrocnemius. For intramuscular injection, 30 μL is close to the limit of volume that can be injected. Therefore, 28-gauge insulin syringes are preferred because, in our experience, they eliminate the loss volume in syringe needles.</li>
<li>After the injection is complete, return the mouse to the recovery cage and monitor it continuously until awake. Allow the animal to recover for several hours and then proceed with the in vivo bioluminescence imaging of the transplanted cells.</li>
</ol>
4. Bioluminescence imaging of ESC-derived endothelial cells in vivo
<ol>
<li>The transplanted ESC-derived endothelials cells were modified to express both the fluc and the eGFP fusion gene under the control of an internal ubiquitin promoter. Therefore, bioluminescence can be used to track the cells within the ischemic hindlimb.</li>
<li>To begin this step, turn on the bioluminescence imaging system and Living Image acquisition software. Then initialize the acquisition system and specify the dimensions of the field of view.</li>
<li>Next, place black matte paper onto the imaging box to absorb background emission.</li>
<li>Once the imaging box is ready, place the mouse into the anesthesia induction chamber containing 1–3% isoflurane in oxygen at output of 1L/min. Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber.</li>
<li>Remove the hair from both hindlimbs using an electric shaver as necessary.</li>
<li>Inject 10 μL of D-luciferin per gram of body weight into the peritoneum. The D-luciferin is prepared in advance into filtered stock solutions of 15 mg/mL in PBS.</li>
<li>Once the luciferin is injected, place the animal into the imaging box over the black paper in the supine position, connected to a continuous flow of isoflurane.</li>
<li>Placing animal into imaging box and connecting to isoflurane.</li>
<li>Start to acquire images for 10–60 seconds in order to determine an optimal exposure time for which the image is not saturated. If the image becomes saturated, reduce the exposure time. If the bioluminescence signal is very weak, increase the exposure time.</li>
<li>Using the optimal exposure time, continue acquiring pictures every 1–3 minutes until the signal reaches the maximum and then fades. When acquisition is complete, save the file.</li>
<li>To analyze the data, select regions of interest (ROIs) that cover the injection site. As a negative control, a similar ROI can be selected for the non-operated leg. Using the software, measure the total radiance, which is expressed in units of photons/seconds/cm2/steradian (p/s/cm2/sr), for the ROIs for each timepoint. The maximal value should be used in the final data. The data can also be exported to Excel spreadsheet for future use.</li>
<li>Once all the data is acquired, return the animal to the recovery cage and monitor continuously until the animal awakes.</li>
<li>Repeat this procedure to track the cells over time.</li>
<li>At a desired time point, the animal can be euthanized for assessment of tissue function.</li>
</ol>
5. Representative Results
<img src="/files/ftp_upload/old/1034_2009_1_22_4/figure%201.png" alt="Bioluminescent imaging of mouse tumor progression; time-lapse and intensity graph analysis." width="600" height="600" data-altupdatedat="1787630408000" data-alt="Figure 1">
A representative bioluminescence image of transplanted cells in the left ischemic hindlimb is shown in Figure 1. During acquisition of bioluminescence, the intensity will increase with time, and the maximum value obtained during the time course should be reported as the final value.

<ol><li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+model+of+hindlimb+ischemia%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li>
<li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cells+derived+from+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li>
<li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li>
<li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li>
<li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization%5BTitle%5D)+AND+%22J.+Am.+Coll.+Cardiol%22%5BJournal%5D)">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li>
<li>Cook, M. J. The anatomy of the laboratory mouse. , Academic Press. New York. (1976).</li>
<li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioluminescent+indicators+in+living+mammals%5BTitle%5D)+AND+%22Nature+Med%22%5BJournal%5D)">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li>
<li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li>
<li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells%3A+potential+for+cardiovascular+tissue+engineering%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li>
<li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+visualization+of+embryonic+stem+cell+survival%2C+proliferation%2C+and+migration+after+cardiac+delivery%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li>
<li>Wilson, K., Yu, J., Lee, A., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li>
</ol>

ESCs are a promising cell source for the treatment of tissue ischemia because of their plasticity of differentiation and their ability to give rise to cell lineages comprising all three germ layers, including endothelial cells. To overcome the ethical concerns associated with ESCs, induced pluripotent stem cells (iPSCs) may be an alternative pluripotent stem cell source that overcomes the ethical concerns. Besides ESCs, adult stem cells such as endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) can...

<table><tbody><tr><td>Surgical tools</td><td>Tool</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Syringe needle</td><td>Tool</td><td>BD Biosciences</td><td></td><td>28G insulin syringe is preferred</td></tr><tr><td>Phosphate Buffered Saline </td><td>Reagent</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>D-luciferin</td><td>Reagent</td><td>Biosynth International, Inc</td><td></td><td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td></tr><tr><td>IVIS 200 Bioluminescence imaging system and acquisition software </td><td>Equipment</td><td><a href="http://www.perkinelmer.com/Catalog/Category/ID/IVIS%20Series" target="_blank"><img src="/img/new_window.png"> Xenogen Corporation</a></td><td></td><td></td></tr></tbody></table>

embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Peripheral arterial disease. PAD results from a narrowing of the peripheral arteries that supply blood and oxygen to the legs and feet leading to vascular dysfunction. To determine whether or not stem cells can be used to treat PAD, we inject embryonic stem cell derived endothelial cells into the hind limb following the induction of ischemia.Since these cells have been modified to express firefly luciferase, they can be tracked by bioluminescence imaging in order to determine whether they home to the site of injury and have therapeutic effect. Hi, I'm John Cook. I'm professor of medicine here in the division of Cardiovascular medicine at Stanford University.Our laboratory is interested in angiogenesis and vascular regeneration. In this video, two of my postdoctoral fellows, Dr.Nan Wong and Hir Niyama, will introduce you to procedures for therapeutic cell transplantation in heim limb ischemia and non-invasive imaging of cell survival. These procedures includes the following steps, the delivery of embryonic stem cell direct and stereo cells for treatment of unilateral hind limb ischemia and non-invasive tracking of cell harming and survival by biolace imaging image.So let's get started. Begin the procedure by preparing an eight month old SV 1 29 strain mouse that has undergone hind limb ischemia for cell transplantation. To do this, place the mouse into the anesthesia induction chamber containing one to 3%ISO fluorine in 100%oxygen at a flow rate of one liter per minute.Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. Then place the animal in the supine position onto the operating table and connect it to a continuous flow of one to 3%ISO flow rain in 100%Oxygen at a flow rate of one liter per minute.Wipe the skin of the hind limb with three alternating Betadine and alcohol scrubs. Once the skin is cleaned, obtain 1 million murine embryonic stem cell derived endothelial cells in 30 microliters of phosphate buffered saline. PBS load these cells into a 28 gauge needle.When the cells are ready, gently lift and extend the hind limb to better visualize the location of the gastrocnemius muscle. While the leg is extended, insert the needle through the skin into the underlying muscle. Take care not to approach the bone gently and slowly inject the 30 microliter cell mixture into the gastroc anemia.For intramuscular injection, 30 microliter is close to the limit of volume that can be injected. Therefore, 28 gauge insulin syringes are preferred because in our experience they eliminate the loss volume in syringe needles. After the injection is complete, return the mouse to the recovery cage.Allow the animal to recover for several hours, and then proceed with the in vivo bioluminescence imaging of the transplanted cells. The transplanted ESC derived endothelial cells were modified to express both the firefly luciferase gene and the enhanced green fluorescence protein fusion gene under the control of an internal ubiquitin promoter. Therefore, bioluminescence can be used to track the cells within the ischemic hind limb.To begin this step, turn on the bioluminescence imaging system and living image acquisition software. Then initialize the acquisition system. Then specify the dimensions of the field of view.Next place black mat paper onto the imaging box to absorb background emission. Once the imaging box is ready, place the mouse into the anesthesia induction chamber containing one to 3%isof fluorine in oxygen at output of one liter per minute. Leave the mouse in the induction chamber until it is unresponsive to external stimuli.Then remove the animal from the induction chamber. Inject 10 microliters of Lucifer pergram of body weight into the peritoneum. The luciferian is prepared in advance into filtered stock solutions of 15 milligram per milliliter in PBS.Once the luciferian is injected, place the animal into the imaging box over the black paper in the supine position connected to a continuous flow of ISO fluorine. Start to acquire images for 10 to 60 seconds in order to determine in optimal exposure time for which the image is not saturated. If the image becomes saturated, reduce the exposure time.If the bioluminescence signal is very weak, increase the exposure time. Using the optimal exposure time, continue acquiring pictures every one to three minutes until the signal reaches the maximum and then fades. After each acquisition is complete, save the file to analyze the data, select regions of interest or ROIs that cover the injection site as a negative control.A similar ROI can be selected for the non-operated leg. Using the software. Measure the total radiance for the same ROIs for each file.The maximal value should be used in the final data. The data can also be exported to Excel spreadsheet for future use. Once all the data is acquired, return the animal to the recovery cage and monitor until the animal awakes repeat this procedure to track the cells.Over time at a desired time point, the animal can be euthanized for assessment of tissue function. Here is a representative bioluminescence imaging time course of transplanted cells in the left ischemic hind limb at one day after cell injection circled in red is the region of interest ROI where the cells were delivered. The color correlates with the number of transplanted cells where red and yellow color indicate stronger bioluminescence intensity when compared to blue and green color.As you can see, the control limb has no bioluminescence, whereas the limb injected with cells shows strong bioluminescence during the acquisition of bioluminescence. The bioluminescence signal in the ROI will increase with time until reaching a maximum value and then decrease in this figure. The maximum value of bioluminescence as measured by the maximum radiance is obtained at 27 minutes from the start of acquisition.This maximum value should be reported for data analysis. We, we've just shown you how to deliver and track ESC derived in serial cells for treatment of hind limb ischemia. This procedure can be preceded by the induction of hind limb ischemia, which is demonstrated in another job video by ER et all.When doing this procedure, it's important to remember to limit the volume of injected cells in PBS to a maximum of 30 microliters in order to prevent subcutaneous leakage of cells. Also, when acquiring bioluminescence images, avoid saturating the image intensity with exposure intervals that are too long. So that's it.Thanks for watching and good luck with your experiments.

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The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

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Learn to deliver embryonic stem cell-derived endothelial cells for treating hindlimb ischemia using bioluminescence imaging. This method enables non-invasive tracking of cell homing and therapeutic effects in vivo.

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

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