Assessing pluripotency of hES cells: Teratoma Formation
Included here is a protocol based on an original courtesy of Martin Pera that we routinely use to generate teratomas by the inoculation of human ES cells beneath the testis capsules of male SCID mice.
Required Materials:
- 6-8 week old male immunodeficient mice (e.g. Beige SCID, NOD.CB17-Prkdcscid/J).
- Dissecting microscope in a laminar flow hood.
- Anesthetic (e.g. Avertin); 1ml syringes and needles
- Sterile surgical tools (2 pairs fine tipped tweezers, scissors, forceps, sutures)
- hES cells resuspended in PBS+5%FBS (typically >1x106cells/ml)
- Fixative (e.g. Formalin, Histochoice)
- Prepare HESC suspension: dissociate cells by brief trypsonization (2min in 0.05% Trypsin-EDTA), pellet cells by centrifugation (3min at 1100 rpm) and resuspend cells in PBS + 5% FBS. We generally use 1 confluent well of a 6 well dish per animal (for injection of both testicles), resuspending cells from each well in 100uL volume in a 1.5mL tube. Keep cells on ice.
- Anesthetise the animal. Swab the abdomen with alcohol, and, using scissors, make a 1cm longitudinal incision through the skin and abdominal wall just below the level of the origin of the hindlimb.
- Carefully move the mouse onto the stage of the dissecting microscope. Locate the fat pads attached to the testis by looking inside the incision towards the caudal part of the mouse and slightly lateral. Pull on one with tweezers; a vas deferens and testis should follow. Rest the fully exteriorised testis on a piece of sterile gauze. Your hES cells should be ready at this point.
- Using a 30 g needle, poke a small hole in the testis capsule in a region where there are no major vessels. The needle should be inserted into the testis about halfway, so the cells do not escape, and slowly inject cell suspension. You can introduce perhaps 50 l before the testis capsule begins to swell. If a few seminiferous tubules herniate out when the needle is removed, do not worry.
- Return the testis back near its original position inside the abdominal cavity. Exteriorise, inoculate and replace the other testis. Suture the internal incision (2-3 sutures) and close the skin closed using autoclips. Keep the animals warm and ensure the rodent successfully comes out of anesthesisa and make sure you check him the next day.
- Monitor the animal weekly beginning at around three weeks for tumour development. Do this by bringing the testis down into the scrotum and palpating it. Lesions usually become evident by around five weeks.
- Kill the mouse humanely before the tumours exceed dimensions which cause the animal obvious suffering. At autopsy, remove the testis carefully and include it and any unidentifiable surrounding tissue for fixation and processing. Tumours from hES cells often form large cystic structures consisting mainly of fluid; more rarely, they are solid lesions. Place the tumours in fixative.
- Cut multiple sections through the specimen to locate areas of interest. Retain alternative sections for staining with hematoxylin and eosin; the intervening sections can be used for alternative analysis. For instance, the human origin of the cells can be confirmed by immunocytochemistry using species specific monoclonal antibodies against widely expressed structural or mitochondrial proteins. The lesions should contain a variety of tissues, including glandular epithelium, muscle, bone, neural tissue, keratinising epithelium, cartilage, and other cell types. The precise identification of tissue types can be accomplished by immunohistochemistry since the tissue is often immature, but broad classification is usually possible. Teratomas from euploid human ES cells should not contain malignant elements (undifferentiated embryonal carcinoma cells). You may wish to enlist the assistance of a histopathologist (ie. CMHD at TCP) to identify the tissues that are present and score the resulting tumours for presence of all 3 germ layers if you are not experienced in the histological examination of teratomas.
Notes:
- We generally use one male animal for each cell type being evaluated and inject both testicles with the cell suspension from the same syringe. Since there is the potential of cells leaking from the injection site and forming tumours outside the testicle (ie. The abdominal wall) it is prudent to avoid introducing multiple cell types into the same animal.
- Autoclaved materials and instruments must be sterilized in glass bead sterilizer between animals.
- The time period for teratoma formation appears to be influenced by the stem cell type used. While mouse ES and iPS cells generally form tumours within 4 weeks, it often requires 6-8 weeks for human ES and iPS cells to form solid tumours. The time frame for incubation must balance the time required for pluripotent cell differentiation without causing undue pain to the mice.
- This protocol can be modified for injection of cells into the kidney capsule