The overall goal of this procedure is to assay pluripotency by generating teratomas in the testis capsule of immune compromised mice using human pluripotent stem cells or H PSCs. Start by harvesting a culture of the putative H PSCs to be tested for pluripotency. Then perform surgery to expose the mouse testes and inject the cells into the testis capsule.
Place the testes back into their original location and surgically close the mouse back up after six to 12 weeks, examine the resulting tumor by histology for the presence of cells from each germ layer. Ultimately, results can demonstrate that an HPSC line is truly pluripotent by showing that it can differentiate into cells from all three germ layers in an immune compromised mouse. Although new methods for demonstrating pluripotency of human stem cells have recently been reported, the teratoma assay remains the gold standard in the field demonstrating the procedure will be tron, a senior technician in my lab Culture.
The human pluripotent stem cells to co fluency and then dissociate the H PSEs with Accutane enumerate the cells to re spend 1 million HPCs per injection. First shave the abdomen of anesthetized six week old male mice of an immunocompromised mouse strain with clippers. Clean the anterior wall of the mouse abdomen with povidone iodine solution, starting from the center of the abdomen and working clockwise outward.
Then wash the shaved area with 70%ethanol. Repeat the process three times, changing swabs each time. To perform the surgical procedure, transfer the animal to a heating pad inside a sterile hood with sterile surgical scissors.
Make a one centimeter longitudinal incision through the skin and peritoneum just below the level of the hip joint while holding the peritoneum with forceps. Reach down toward the right hip with another pair of sterile forceps and pull out the white fatty tissue along with attached testes. Now fill a one CC tuberculin syringe with the hpsc to be injected Slowly inject 20 to 30 microliters of H PSCs into the center of the testis capsule away from any major blood vessels.
Remove the needle slowly to avoid reflux of the cells using forceps. Transfer the testes and fatty tissue back to their original position in the abdomen. Close up the peritoneum with two or three reabsorbable sutures and close the skin with auto clips.
After surgery, administer analgesia twice daily as needed. Monitor the animal for palpable tumor growth over the course of six to 12 weeks. At approximately five millimeter tumor size, remove the tumor from the sacrificed mouse for documentation.
Photograph the tumor and measure both size and weight. Then cut the tumor into small pieces and fix in 4%paraldehyde solution for histopathology. Typically, a teratoma is observed after six to 12 weeks.
In this example, 1 million human WA oh nine pluripotent stem cells were injected into the testis capsule of an immune compromised mouse. Close examination of the teratoma reveals heterogeneity and many attached cysts. As expected.
Histopathological analyses indicate the presence of cells from each of the three germ layers. While attempting this procedure, it's important to remember to include a positive control cell line that you know is pretty potent to make sure that the assay was done correctly. After watching this video, you should have a good idea of how to determine whether or not a human stem cell line is pluripotent.
