In this video, two related techniques are demonstrated. The first is transplantation of spermatogonia stem cells or SSC transplantation, and the second is test these tissues.Xenograft. SSC transplantation is used to generate spermatogenesis from a donor's SSCs in the testes of a recipient.
This is accomplished by preparing a cell suspension from donor testes or cultured cells containing SSCs from a donor with an identifiable transgenic marker such as GFP or LAC Z.Next, the testes of the recipient male are exteriorized. The recipient has been treated so his testes are depleted of germ cells. The EENT ducks of the recipient male are then isolated and injected with donor cells that have been mixed with taan.
Blue dye. Ultimately colonization of the seminiferous epithelium of the recipient. Seminiferous tubules by donor derived germ cells is evident two to three months after transplantation observed in this example by xal staining of the recipient testes demonstrating the colonization of lack Z expressing donor cells.
The second technique to be demonstrated in this video involves testis tissue xenograft. Testis tissue rafting is used to introduce testis development and spermatogenesis from a donor species by transplanting testis fragments from immature donors into deficient mouse hosts. This is accomplished by first preparing suitable tissue fragments for transplantation from the testes of a large donor species such as a pig as in the example shown here.
The next step is to castrate an immunodeficient adult recipient mouse and then transplant the donor testes fragments under the dorsal skin of the recipient mouse. The recipient mice function as in vivo incubators, allowing full development of the testes xenografts within six to seven months depending on the donor species. Speral stem cell transplantation was first developed by Ralph Brister and his colleagues at the University of Pennsylvania and first reported in 1994.
It since has found widespread application and currently represents the gold standard bioassay in the field of germline stem cell biology to help answer key questions in the field of germline stem cell biology, such as elucidating signaling pathways that govern stem cell self-renew. We first had the idea for xenotransplantation when we recognize that transplantation of isolated spermatogonia stem cells from non rodent donors, for example, pigs, primates, or humans to a mouse testis, did not result in complete donor derived spermatogenesis. We established that preserving the microenvironment of the testis by transplanting small fragments of testis tissue rather than isolated germ cells can overcome this limitation and lead to complete donor derived spermatogenesis from a phylogenetically distanced species in a mouse host.
This was first reported by us in 2002. The germ cell transplantation protocol requires the preparation of the cell suspension to be injected. Consult the written manuscript for preparation details.
Begin the transplantation procedure by anesthetizing the recipient mouse and ensuring sufficient anesthetic depth by making sure that it does not display a pain reflex. Then position the animal dorsal recumbent and surgically prepare the abdominal area. Make an approximately one centimeter long midline incision to expose the abdominal wall.
Lift the wall using forceps at the point of the white line to avoid accidentally injuring abdominal organs. Then make a half centimeter incision to expose the peritoneal cavity with the help of fine forceps. Exteriorize the testis by pulling the fat pad attached the testis and the epididymus, and place these structures on a sterile index card.
Now add trian blue dye to the cell suspension. Then load the cell suspension into a polyethylene tubing connected to a one milliliter syringe and attach the pulled pipette into the tubing. Gently force the cell suspension into the pipette by applying pressure to the syringe.
Now we are going to prefer the most critical step for SCC transplantation, which is the identification and cannulation of the ENT ducts. Using a dissecting microscope, identify the efferent ducts between the testes and the EPIs and gently remove the fat tissue around the ducts. Work carefully as the ducks and the membrane around them are translucent.
Stabilize the ducts by cupping them with a sterile triangular piece of plastic. Then carefully insert the injection pipette into a duct in the bundle of a ferrin ducts. Gently thread the pipette a few millimeters toward the testis.
Keeping the injection pipette still slowly inject the cells as the ret testis and ferous tubules begin to fill, sustain a steady flow until almost all of the tubules visible at the surface tubules have been filled. Do not overfill or the testis will become ischemic. Now return the testis to the abdominal cavity and repeat the procedure on the contralateral testis.
When completed, close the abdominal wall with six oh silk suture and close the skin with metal wound clips. Post-surgery, monitor the mouse on a warming pad until it is recovered sufficiently from anesthesia to be returned to its home cage age. The second procedure to be demonstrated the testis tissue xenotransplantation involves the use of testis biopsies or intact testes from donors.
In the case of biopsies, wash the testis fragments two or three times in ice cold culture medium containing antibiotics. After each wash, spin down the suspension and bring up the pellet. In fresh solution, transfer the wash tissue to a clean dish and cut the biopsies into smaller fragments for transplantation.
Using curved forceps and a scalpel blade intact testes require more preparation. Begin by washing each testes and ice cold PBS containing antibiotics two or three times. Then transfer the clean testes into a clean dish with PBS.
Next, remove the tunic of vaginalis by making an incision along the surface and extrude the testes. Remove all annex structures such as matic, cord epididymus, and connective tissue. Wash the extracted testes once in cold PBS and transfer them to a culture dish with PBS.
Next, carefully remove the tunica albuginea using a scalpel blade in scissors. If the testes is very small, the tunica can be removed by squeezing the testicular tissue out of the tunica through a small incision in a small culture dish filled with ice. Cold medium, gradually dice the testes into small pieces as described previously.
Finally, select tissue fragments to be xenograft in and isolate them in culture dishes with ice cold culture medium until they're needed. Anesthetize a mouse as previously described. Then with the mouse on a warming pad.
Prepare the sterile surgical field by clipping the hair on the abdomen and back. Wipe down the abdomen with 70%ethanol and Betadine solution. To perform the castration, make a one half to one centimeter ventral midline skin incision to expose the abdominal wall.
Then cut through the abdominal wall to expose the abdominal cavity. Carefully expose the testis, the testicular artery and epididymus as previously described. Then detach the tail of the EPIs from the gubernaculum by blunt dissection.
Next, ligate the testicular artery in the VA deference together with the blood vessel using silk sutures. Then section the ligated structures by cutting between the testes and the ligature. After repeating the procedure for the second testis, suture the abdominal wall with one or two surgical stitches and close the skin incision with one or two micc clips to implant the ectopic xenografts.
Position a castrated anesthetized mouse in ventral recumbent and prepare a sterile surgical field on its back. For each graft to be inserted, make a half centimeter long skin incision on each side of the midline using forceps to hold a border of the skin incision. Use scissors to make a five to 10 millimeter deep subcutaneous cavity.
Tease apart the connective tissue to make a space deep enough for the graft such that it will not be caught by the closure clip. Using fine forceps. Place a piece of the testis tissue deep into the subcutaneous cavity holding the border of the skin incision with an iris forceps.
Close each skin incision with one mic clip. Then allow the mouse to recover from anesthesia on a heating pad. Once recovered, transfer the mouse to its home cage and continue to monitor it until it is fully recovered.
Transplanted Donor spermatogonia stem cells expressing a lae transgene were found to colonize the recipient's testis two to three month post transplantation iCal. Staining revealed distinctive blue segments of the seminiferous tubules of the recipient. A well-established colony has a long, dark blue stretch of completely filled segments with two or more layers of blue cells closer to the center at either end of the colony.
A network of single paired or small groups of cells is also apparent. A cross section of the dark blue seminiferous tubule region revealed, well-established and well-organized Perato agenesis with blue germ cells at various differentiation stages. In the case of testis tissues, xenograft fragments of immature donor testis were transplanted under the dorsal skin of immunodeficient mice and were able to survive and respond to mouse gonadotropins.
And as a result, testis tissue underwent complete development, including formation of fertilization competent sperm. Once testis xenografts were collected, they were used to obtain sperm for exy and embryo production. Sperm moron stem cell transplantation advances the field of stem cell biology through the study of stem cell niche interactions in the testes and the development of culture conditions to expand sperm medicon stem cells in vitro after the development of testis tissue xenograft.
The technique paved the way for researchers in the field of male reproductive biology to study aspects of spermatogenesis reproductive toxicology and the preservation of genetic material from immature males in a variety of donor species ranging from mice to men.
