in vitroおよびヒト胚性幹細胞のin vivoでの生物発光レポーター遺伝子イメージングの

In vivo, teratoma formation is the gold standard for determining a cell's pluripotency or ability to form all three germ layers. Teratomas are also a big obstacle for stem cell transplantation, and currently little is known about their formation in growth kinetics. Once undifferentiated cells are implanted into the host environment, the development of bioluminescent reporter, gene transduction and ultra-sensitive charge coupled device or CCD photography has enabled non-invasive repetitive assessment of cell location migration, proliferation, and differentiation in vivo.

In this video, we will demonstrate a detailed protocol for effectively tracking transplanted human embryonic stem cell proliferation in a living mouse. Hi, I'm Kit Wilson from the laboratory of Joseph Wu in the Departments of Medicine and Radiology at Stanford University. Today we'll show you procedures for imaging human embryonic stem cells, both in vitro and after transplantation into a living mouse.

These procedures include the following steps in vitro bioluminescence imaging of human embryonic stem cells, mouse preparation and stem cell injection for in vivo imaging and in vivo bioluminescence imaging of transplanted cells. So let's get started by reviewing some of the key components of these procedures. In order to perform bioluminescence imaging of human embryonic stem cells, you first need to obtain cells that stably express EL luciferase reporter genes such as firefly luciferase, driven by a constitutive promoter like ubiquitin or eef one alpha culture, your EL Luciferase positive human embryonic stem cells in feeder free conditions.

We typically follow the standard Y cell protocol and use six well plates coated in matrigel using either condition media or commercial feeder free media. For detecting the luciferase positive cells, you'll need to have the reporter probe d Lucifer, already prepared to do this. Aliquot Lucifer in one to 1.5 milliliter preparations at a final concentration of 45 milligrams per milliliter.

Keep the aliquots at minus 20 degrees Celsius when not in use and avoid exposure to light by covering with paper towel when not in storage. To visualize the luciferase positive cells, we'll use two different imaging systems. For the in vitro procedure, we'll use a sterile image system, the Xeno IVUS 50, and for the in vivo, we'll head over to Stanford Center for in vivo imaging to use the Xeno IVUS 200.

This system includes an integrated isof lorrine apparatus and induction chamber for temporary anesthesia of small animals. Okay, I've got myself. Let's move on to the in vitro bioluminescence imaging.

For in vitro bioluminescence imaging, it is important to maintain sterile conditions. Therefore, the imaging system should be sterile, preferably in a cell culture room. Before imaging, remove the cell media and add just enough PBS to cover the cells.

For example, add one milliliter of PBS to each well of a six well plate containing your human embryonic stem cell cultures. The ratio of derin to PBS should be one to 100. So add 10 microliters of thaw derin to each well of the six well plate.

Remember to filter the derin before adding to your cultures. After waiting one minute, place the plate into the chamber, then take an image using the exposure time of 10 seconds, adjust the exposure time. If the signal is saturated or too weak, the bioluminescence signal reflects cell number, so quantitation assays can be performed that correlate signal with different cell numbers.

These are images taken from a typical quantitation assay. In the upper left is a bright field of some human embryonic stem cell colonies. We know these cells carry our reporter gene since they fluoresce green after fact.

Sorting the blue color is dappy staining. The bottom panel shows cultural wells with increasing numbers of cells. Cell numbers correlate with bioluminescence signal as seen in this graph.

This correlation gives us a semi-quantitative assay for tracking cell proliferation in vivo. Okay, that's the in vitro procedure. Now let's move on to the in vivo bioluminescence imaging.

To image teratoma formation in vivo, we will inject our human embryonic stem cells that express the firefly luciferase reporter gene into the back of a skid mouse and take bioluminescent images with the IVU system. When you're ready to transplant the cells, use disc space or collagenase to loosen the cells wash several times and suspend them in a one-to-one mixture of growth factor, reduced matrigel and dmm. Typically, we first mix the cells with chilled DMM and then add in the matrigel.

For each subcutaneous site, we inject 200, 000 cells suspended in 50 to 100 microliter volume of the matrigel DMM mixture. The cell number can be adjusted depending on your application. Remember to keep everything on ice prior to injection.

Once the cells are ready, we anesthetize the mouse by placing it in the induction box. With iso fluorine flow turned on. After about one minute, the mouse is anesthetized and can be placed on the operating table with continuous iso fluorine.

Because for naturally auto fluoresces and may obscure the bioluminescent image, we will need to remove the hair from the backside of the mouse. The easiest way to do this is to use an electric shaver, but you can also use hair removal gels, wipe with alcohol to sterilize the skin afterwards. Next, draw up your a hundred microliter cell suspension in a syringe 23 to 27 gauge needles work best since they will not clog up with the cells.

Now, inject the cells under the skin into the mouse's back because the skin is quite loose. Use your thumb and forefinger to pinch and stretch out the area you want to inject. Inject just under the skin taking care not to puncture too deeply.

Also, try to keep the needle from sliding out of the injection site while depressing the plunger. This will prevent creating a hole in the skin through which the cells can leak. If you try to inject again after the cells are injected, wait a few hours to allow the mouse to wake up and run around before putting it back to sleep.

For bioluminescence imaging, doing so avoids isoflurane toxicity. Okay, now we're ready to proceed with in vivo bioluminescence imaging of the injected cells. For the following imaging studies, we'll be using a mouse that has already begun to show formation of a teratoma for whole animal bioluminescence imaging.

We do an intraperitoneal injection of Lucifer at 375 milligrams per kilogram body weight. Therefore, we need to weigh the anesthetized animal to calculate the dosage, inject the deed lucifer into the peritoneum, just off the midline, draw back on the needle and make sure there is no blood, which would be an indication that you've damaged in an internal organ. If the mouse begins to wake up, just place it back in the induction chamber and wait a minute or two.

Prepare for imaging by placing matte black paper in the imaging box to help absorb any light, not emitted by the human embryonic stem cells. For mice, the maximum bioluminescence signal usually occurs 15 to 40 minutes after injection. So after 10 to 15 minutes, place the mouse backside up in the imaging chamber with iso fluorine.

Start with an exposure time of 10. If the signal is saturated, try decreasing the exposure time. If too weak, then increase the exposure.

Once the signal exposure time has been optimized for your mouse, begin taking the images every minute until the signal reaches a maximum. This is best done by using the imaging software to select regions of interest that cover your injection sites. By monitoring the signal intensity at each region of interest, you can easily determine when the signal begins to decrease, indicating that the maximum signal intensity has been reached.

We take the average of three successive images that occur around the maximum and use that as our final value. When you are satisfied with your images, remove the mouse from isof fluorine and allow it to wake up in its cage. Usually the mouse should wake up within 15 minutes.

After the desired time course of bioluminescence images is acquired. The animal may be sacrificed in tissue sections used for histology. Okay, now that we're all done with the in vivo imaging procedures, we can take a closer look at our results and also look at some previous studies from our lab.

Here is a bioluminescence image of three mice with subcutaneous teratomas. The bioluminescence signal data has been overlaid on a black and white photo of the mice done automatically for us by the imaging software. This is an example of the type of longitudinal imaging that's possible with firefly luciferase.

In this case, we injected human embryonic stem cells directly into the heart after the initial injection. Most cells die during the following week as shown by a decrease in bioluminescence over the heart. Note, the black arrow.

The human embryonic stem cells that survive transplantation ultimately form a teratoma in the heart that can be visualized as a rapidly increasing signal starting at around day 14. You'll notice that at day seven there's a second region of bioluminescence indicated by the red arrow that shows extra cardiac leakage of human embryonic stem cells into the abdomen. This is very common.

We've just shown you how to acquire bioluminescence images of human embryonic stem cells that stably express a luciferase reporter gene, both in vitro and after transplantation to a living mouse. This method allows for repetitive imaging and monitoring of cell proliferation without the need to sacrifice large numbers of animals for histology, which may be inherently inaccurate for cell quantitation. When doing this procedure in a mouse, it's important to remember to use the correct concentration of delucci and to avoid saturating bioluminescence image with exposure intervals that are too long.

So that's it. Thanks for watching and good luck with your experiments.