The overall goal of this procedure is to isolate rodent urogenital, sinus mesenchyme or UGSM from rodent embryos and implant tissue mixtures into the renal capsule that can form human prostate epithelium. This is accomplished by first isolating the urogenital sinus from mouse or rat embryos. Next, the urogenital sinus mesenchyme is separated from the epithelium.
Then single cell suspensions of urogenital sinus mesenchyme are combined with pluripotent stem cells. Finally, the cell mixture is implanted under the renal capsule of a host mouse. Ultimately, results can be obtained that show human prostate epithelium formed on the mouse kidney through validation of human specific PSA expression.
Though this method can provide insight into prostate function and disease initiation, it can also be applied to other systems such as xenograft of human prostate tumors and induction of weekly tumorogenic cell lines to form tumors. After euthanizing a timed pregnant mouse or rat and isolating the embryos, separate them from the amniotic sack, cut the umbilical cord and transfer them into a 50 milliliter tube containing ice cold hanks. Balance salt solution and keep on ice.
Place an embryo into a 100 millimeter Petri dish and use scissors to bisect it at the level of the bolus. Remove the stomach and small intestine and then locate the ovaries in the female embryo, which are found at the coddle poles of the kidneys or the testis in the male located at the level of the bladder. Under a dissection scope.
Use Venice scissors to cut the abdominal wall to expose the bladder and posterior wall of the abdomen. Free the upper genital tract from the attachments and free the umbilical cord. Cut the pubic bone and with dumont fine tip forceps.
Bluntly, separate it from the anterior wall of the urogenital sinus. Separate the posterior wall of the urogenital sinus from the rectum and cut the urogenital sinus with the bladder at the lower end. Then separate the bladder from the urogenital sinus.
Store the urogenital sinus and block in ice cold HBSS. Transfer the urogenital sinus to a 50 milliliter tube containing 1%trypsin in HBSS without calcium and magnesium and incubate at four degrees Celsius for 30 minutes. After the incubation, remove the trypsin and add 10%FBS and DM A MF 12 medium to neutralize the digestion.
Next, use a needle or fine tip forceps To carefully tease the sticky mesenchymal sleeve from the epithelial tube. Maintaining the epithelial tube intact reduces the chances of epithelial cell contamination of the mesenchyme. This can result in rodent glands forming along with the stem cell derived human glandular epithelium.
Transfer the UGSM into a new 50 milliliter tube containing DMM F 12 medium containing 10%FBS 1%pen strep, 1%non-essential amino acids and one nanomolar. R 1 8 81 Incubate the tube at 37 degrees Celsius with 5%CO2 overnight. The next day centrifuge the tissue at 200 G.Discard the supernatant and use PBS to wash the pellet.
After removing the PBS resuspend the tissue in 0.2%Collagenase in D-M-A-M-F 12 and incubate at 37 degrees Celsius with gentle rocking for one hour vigorously vortex the digestion tissue three times until it becomes a homogeneous single cell mixture and neutralize the collagenase by adding D-M-A-M-F 12 medium with 10%FBS 1%pen strep, 1%non-essential amino acids and one nanomolar R 1 8 81. Wash the cells three times by pelleting them at 200 G and Resus suspending them in HBSS. Finally, resus, suspend the mesenchymal cells in DMM F 12 and use a hemo cytometer to count them to carry out the transplantation.
After preparing a sterile surgical area and anesthetizing a male athymic nude mouse with an IP injection of ketamine xylazine, according to the text protocol, perform a toe pinch to confirm level of sedation surgically prepare the mouse by shaving the surgical site if necessary, and cleansing it with three alternating wipes of 70%alcohol followed by Betadine solution. After applying eye ointment, make a one centimeter longitudinal incision on the back and continue to cut the abdominal wall with a 0.5 centimeter longitudinal incision. Gently squeeze the kidney out of the abdomen and use drops of sterile saline to keep it moist.
Next, using a 27 gauge syringe needle, gently puncture the renal capsule to create an injection site for the cell mixture. Then fill a 28 gauge syringe needle with 10 microliters of cell mixture and slowly insert it into the puncture site, penetrating the kidney parenchyma. To reach an area underneath the renal capsule.
Inject 10 microliters of the cellular mixture to form a bolus on the kidney underneath the renal capsule and withdraw the needle. Return the kidney to the abdominal cavity and secure the muscular layer of the peritoneum with four aught nylon sutures. Close the skin with either sutures or staples and use sterile saline to clean any blood from the skin.
Shown here is an ultrasound image of a growing xenograft eight weeks after implantation. The circled area represents the xenograft on the kidney surface. These images demonstrate xenograft growth on the kidney surface.
After 12 weeks, human ES cells implanted in the absence of urogenital sinus, mesenchyme will form a large teratomas, whereas urogenital sinus mesenchyme implanted alone will fail to form any discernible structure. Shown in the center. Panels of this figure are representative immunohistochemical stainings of stem cell derived human prostate epithelium versus adult human prostate tissue.
As shown here on the left, the glands contain an AR positive luminal epithelial layer. That is also PSA positive, a P 63 positive basal epithelial cell layer and rare chromogranin. A neuroendocrine cells, the glands are not cancer as shown by negative A-M-A-C-R staining upon host castration.
The AR PSA positive luminal cells are no longer present demonstrating characteristic androgen dependency. While attempting this procedure, it's important to remember to take great care in separating the urogenital sinus mesenchyme from the epithelium. As contaminating urogenital sinus epithelium will form glands and potentially confound your results.
