The overall goal of this procedure is to differentiate human embryonic stem cells in vivo and determine their tissue specific fates. This is accomplished by first creating a pellet from genetically tagged cells of interest. The second step of the procedure is to surgically place the pellet under the kidney capsule of an immunocompromised mouse.
The third step of the procedure is to allow the cell pellet to differentiate for eight to 12 weeks beneath the capsule of the mouse kidney. The final step of the procedure is to harvest the resulting teratoma, fix it formalin, and analyze the fate of tagged cells by immunohistochemistry. Ultimately, results can be obtained that show the tagged cells developing into one or more of the three germ layers through histology and immunohistochemistry of the resulting teratomas.
Hi, I'm Carissa Ritner from the laboratory of Dr.Harold Bernstein in the Cardiovascular Research Institute at the University of California in San Francisco. Today we will show you a procedure for developing teratomas via kidney capsule engraftment. We use this procedure in our laboratory to study the differentiation of embryonic stem cells, both to ensure that each line we work with can develop into each of the three germ layers and attract the fate of genetically tagged stem cells in vivo.
All of the procedures we will show you are done with the approval of the UCSF Institutional Animal Care and Use Committee. Also, all work with human embryonic stem cells is performed with approval from the UCSF Stem Cell Research Oversight Committee. So let's get started.
To prepare human embryonic stem cell cultures, please refer to the text portion of this protocol. For notes in a lamina flow hood, treat the rock inhibitor treated human embryonic stem cell cultures with one milligram per milliliter of collagenase four incubate for five minutes at 37 degrees Celsius and 5%carbon dioxide aspirate the collagenase four from the, well. Replace it with one milliliter of room temperature DPBS supplemented with 10%penicillin streptomycin.
Use a flathead cell lifter to scrape the colonies from the plate and transfer 0.5 milliliters of the cell suspension to each of two micro centrifuge tubes. Rinse the well with one milliliter of DPBS and transfer 0.5 milliliters of the rinse to each tube pulse. Spin the tubes for 20 seconds at 8, 000 times gravity aspirate the supinate and resuspend each pellet in 500 microliters of fresh DPBS at 100 microliters of one milligram per milliliter.
Fasi vulgaris lectin in DPBS to each tube to help bind the cells together for transplantation. Incubate the cell suspension at room temperature for five minutes. Spin the cells at 8, 000 times gravity for two minutes to even out the pellet.
Rotate the tubes 180 degrees within their rotor wells. Then spin again at 8, 000 times. Gravity for two minutes.
Carefully aspirate the SUP nasin, leaving the pellets intact to dislodge the pellets from the tube. Gently pipette 200 microliters of KSR medium next to the edge of the pellet until it slowly lifts up. Once the pellet is dislodged, immediately pull the media into a 0.4 micrometer milli cell.
Insert in a well contain three milliliters of KSR.Medium. Incubate for two hours to overnight at 37 degrees Celsius and 5%carbon dioxide. Place an anesthetized immunodeficient mouse on a heating pad to prevent hypothermia.
Use operating scissors and iris forceps to make a 2.5 centimeter incision through the skin parallel to the spine. But just off center. Make a smaller incision through the muscle just below the ribs and approximately one centimeter from the spine.
The incision should be large enough to pull the kidney through, but small enough that the kidney will not slip back into the abdominal cavity without force. Using a pasta pipette that has been pulled into the shape of a blunt hook, gently pull the kidney through the muscle incision. Periodically wet the kidney with sterile DPBS to prevent the fragile capsule from drying out with fine forceps.
Gently pull the capsule away from the kidney. Use Venice Spring Scissors to make a two millimeter incision in the capsule. At the center of the kidney, insert a pasta pipette that has been pulled to a very fine blunted probe in between the capsule in the kidney and wiggle gently to make a small pocket repeat on the other side of the kidney so that there are two pockets.
One on each pole of the kidney. Using a large bore pipette tip on a disposable transfer. Pipette lift a single pellet from the milli cell insert.
Take as little medium as possible as excess medium can interfere with the pellet transfer. Use forceps to hold up the edge of the pocket on the kidney. Gently place the pellet next to the opening and use the pulled pipette probe to push it into the pocket towards one pole of the kidney.
Place a second pellet into the pocket towards the opposite pole. Carefully place the capsule back down onto the pellets. No sutures are necessary to close the capsule.
Use a hooked pipette to gently push the kidney back into the abdominal cavity. The muscle incision should be small enough that it can be sealed with a single silk suture. Close the skin with a series of four to five interrupted stitches and allow the animal to recover from surgery before returning it to the animal facility eight to 12 weeks after surgery.
Remove the kidney teratoma and any surrounding cysts from the abdominal cavity as a block of tissue. Place the entire block of excise tissue into a 50 milliliter polypropylene tube containing 10%buffered formula in. Allow the teratoma to fix at four degrees Celsius overnight.
Remove the teratoma from the tube and place it on a mat. Use a scalpel to dissect away any cysts and as much kidney as possible without damaging the teratoma itself. Larger teratomas may be cut in half to facilitate dissection and embedding.
Process the teratoma using standard paraffin fixation technique and stain mount sections on slides for analysis and imaging. Refer to the notes in the text portion of the protocol for details. Here we will show you tissue sections from a typical teratoma.
This teratoma was stained with hemat and eoin to identify embryonic tissues, representative of all three germ layers and shows an nascent neural tube structure representative of vector derm. This section provides another example of vector derm, in this case primitive squamous epithelium. Here, cartilage surrounded by a capsule of condensed mesenchyme represents tissues derived from embryonic mesoderm.
Finally, this section showing a glandular intestinal structure demonstrates end dermal tissue within the teratoma to map the fate of genetically tagged stem cells, teratomas were seeded with a mixture of wild type and CD 1 33 positive human embryonic stem cells. The CD 1 33 positive derived cells indicated by arrows were observed in tissues arising from embryonic heder, specifically neuro epithelium and nascent neural tube-like structures. I am Dr.Harold Bernstein.
Carissa has just shown you how to form teratomas from human embryonic stem cell cultures to provide an in vivo development assay. When doing this procedure, it's important to remember to be careful with your cells and watch your aseptic technique. So that's it.
Thanks for watching and good luck with your experiments.
