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.
