Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Ethics statement: all experiments were performed according to the Pasteur Institute Ethic Charter, approved by the French Agriculture Ministry, and to the EU guidelines. A manipulator with training on small rodent surgery, certified by the French Ministry of Agriculture, performs all surgical interventions.
NOTE: See in annex Table 1 showing the 5-step plan procedure.

1. Selection of the Embryos

1. Use 36 C57BL/6 CD45.2 females, 12 females CD45.1, 12 C57BL/6 CD45.1 males and 12 C57BL/6 CD45.2 males. Crossing the females with either male genotype will produce embryos CD45.1/2 and CD45.1 used as a source of TSP or CD45.2 used as a source of recipient thymic lobes. House all males individually.
2. To obtain embryos for E18 TSP isolation, place 2 females in a cage with one male CD45.1 at 6 pm, 21 days before the grafting experiment. Check the presence of a vaginal plug the next day, before noon. Separate the plugged females.
3. To obtain E14 embryos to be colonized by TSP, repeat the procedure above (1.2), using males CD45.2, 17 days before the grafting experiment.
4. To obtain embryos to isolate E13 TSP, repeat the procedure above (1.2), using males C57BL/6 CD45.1, 16 days before the grafting experiment.

2. Dissection of the Embryos Under a Horizontal Laminar Flow Hood

NOTE: Two days before the grafting experiment.

1. Sacrifice the pregnant females by cervical dislocation or by progressive administration of CO₂ gas during at least 5 min, in a saturating closed chamber. Ensure death by definitive arrest of cardio-respiratory movements. Wet the abdomen with 70% ethanol.
2. Make a longitudinal incision in the skin at the midline abdomen and open it by pulling the skin apart. Open the peritoneum with forceps and scissors without touching the digestive tract. Pull out the bifid uterus and separate it from the vagina with the scissors.
3. Place the uterus in a 90 x 15 mm Petri dish containing 40-50 ml DPBS and cut transversally between each embryo’s decidua.
4. With the pair of fine tip scissors and a fine forceps remove the muscle membrane of the uterus, take out the embryos by cutting between the placenta and the yolk sac, and remove the amnios, which is retaining the embryos.
5. Place the embryos in a Petri dish 90 x 15 mm containing HBSS + 1% fetal calf serum (FCS). For embryos older than E15, remove the heads with a pair of scissors before any further dissection.

3. Isolation of the Thymus

1. Under the binocular magnifying lens, place the embryo, lying in supine position (ventral view), on a wet gauze bedding.
2. Insert one forceps longitudinally along the cartilage of the sternum, pinch and open the thoracic grid. With curved forceps, pull the thoracic wall aside to visualize the thymic lobes on each side of the trachea.
3. Carefully hold each lobe by pinching underneath with a fine forceps and place them in a Petri dish containing 1 ml HBSS + 1% FCS. Isolate the lobes and remove surrounding connective tissue with two 1 ml syringes + 26 G ⅜" needles, without damaging the capsule.
4. Wash the lobes in 3 ml of HBSS + 1% FCS to eliminate blood cells.
   NOTE: Before E15, thymic lobes are located on each side of the trachea and later fuse in the middle to constitute a bi-lobar organ.

4. Cell Suspensions

1. Tease apart the lobes under the binocular magnifying lens, with two 26 G needles mounted on 1 ml syringes, in HBSS + 1% FCS. Filter the cell suspension through a nylon mesh.

5. Staining with Fluorescent Antibodies and Cell Sorting

NOTE: All antibodies are previously titrated to obtain optimal definition of the populations (the titration can vary with the antibody batch).

1. Incubate the thymocytes (E13 or E18) for 15-30 min at 4 °C with a 100 μl of a cocktail of biotinylated antibodies to stain lineage positive cells (anti-CD19, anti-Gr1, anti-Ter119, anti-NK1.1, anti- CD11c, anti-CD3, anti-CD4, anti-CD8, anti-CD25).
2. To wash away excess of antibodies spin down the tubes with 4 ml HBSS + 1% FCS at 280 x g for 7 min, eliminate the supernatant and re-suspending the pellet in fresh HBSS + 1% FCS. Repeat the centrifugation.
3. Incubate the E18 biotin-labeled cells with streptavidin microbeads during 15 min at 4 °C and wash the cells twice in HBSS 1% FCS. Re-suspend the cells in 2 ml HBSS + 1% FCS. Most E13 thymocytes are lineage negative and therefore do not need MACS enrichment prior to cell sorting.
4. Pass the cells on the LS column, according to the manufacturer’s instructions. When all the cell suspension entered the column add 2 ml HBSS + 1% FCS. Recover the 4 ml of the column flow through and centrifuge the cells for 7 min at 280 x g.
5. Re-suspend the pellet of either depleted E18 or total E13 thymocytes in 50 μl of the TSP antibody mix (anti-CD117 APC, anti-CD135 PE, anti-CD127 PECy7, anti-CD24 FITC, anti-CD44 APCCy7 and streptavidin Pacific Blue) and incubate for 20 min at 4 °C in the dark.
6. Wash the cells with 4 ml HBSS + 1% FCS and re-suspend the cells in 1 ml HBSS + 1% FCS with propidium iodide.
7. Sort the TSPs Lin^–CD44⁺CD117⁺CD24^lowCD127⁺CD135⁺ cells in a FACS (with 4 lasers) onto a 1.5 ml tubes containing 200 μl complete medium + 20% FCS. Gate first the cells according to their size and granularity on the FSC and SSC channels. Eliminate dead cells (staining with propidium iodide), gate out doublets and score the fluorescence in exponential scales. Around 100 or 600 TSPs can be obtained from one E13 or one E18 thymus, respectively.

6. Colonization of E14 Thymic Lobes with Progenitors: Hanging Drop Technique

1. Irradiate (30 Grays = 30 Gy) E14 thymic lobes from CD45.2 embryos, 3 hr before culture.
   NOTE: E14 or E15 thymic lobes have been successfully used as recipient of T cell progenitors. Using recipient thymic lobes of later gestational days results in premature necrosis due to the bigger size of the organ while thymic lobes at earlier gestational days develop poorly, probably due to incomplete development of the epithelial cells.
2. Centrifuge sorted TSPs and re-suspend the cells in culture medium (OPTI-MEM complete medium with 10% FCS, penicillin (50 units/ml), streptomycin (50 µg/ml) and 2β-mercaptoethanol (50 µM)) such that 35 μl contain 500 TSP.
3. Place a 35 μl drop of cell suspension every other well of a 60 well Terasaki plate.
   NOTE: 35 μl drops can fuse if they are placed in contiguous wells.
4. Place one irradiated lobe on the surface of each drop. Close the Terasaki plate and turn the plate upside down to let the cells reach the apical pole of the drop by gravity.
5. Hit gently the upper side of the inverted plate to force the lobes to slide to the apical edge of the drop. Incubate the inverted Terasaki plate in a humidified incubator for 48 hr at 37 °C + 5% CO₂. After colonization, either culture E14 thymi in FTOC⁷, or graft under the kidney capsule of a recipient adult mouse ⁸.

7. Fetal Thymic Organ Culture

1. Place 3 ml of complete medium on a 35 mm Petri dish. Place an isopore membrane filter floating on the medium. Place the reconstituted lobes on the edge of the membrane with a forceps (no more than 8 lobes on one membrane).
2. Place the Petri dishes containing the thymic lobes inside a 90 mm Petri dish, together with a 35 mm dish, without cover, containing 2 ml of water, to ensure optimal humidity. Incubate 12 days at 37 °C + 5% CO₂.

8. Grafts Under the Kidney Capsule Under Sterile Conditions

NOTE: The kidney parenchyma is surrounded by connective tissue forming a capsule. The sub-capsular region is particularly rich in blood and lymphatic vessels thereby providing a suitable environment for the development of grafts (e.g. thymic lobes, pancreatic islets or newborn hearts). Grafts are usually done on the left kidney because it is more accessible than the right kidney. CD3^-/- male mice were used as recipients thus avoiding graft versus host reaction due to minor histocompatibility antigens linked to the Y chromosome because sex determination before E15 is not easily done.

1. Sterilize the instruments and wear sterile gloves along the surgery. Anesthetize the mouse by intra peritoneal injection of a solution of Ketamine 10 mg/ml + Xylazine 1 mg/ml diluted in PBS: (50 to 100 μl per 10 g body weight), using a 1 ml syringe. Verify anesthesia by checking lack of interdigital reflexes. Place a drop of hydro-Optimune gel on each eye to prevent dryness during anesthesia.
   NOTE: The solution should be prepared extemporaneously and warmed at room temperature before injection. The anesthesia lasts at least 20 min. The degree of anesthesia should be controlled all along the surgery. The anesthesia can be prolonged by intra peritoneal injection of half dose every 20 min.
2. Place the mouse on its right side, under a horizontal lamina flow hood. Sterilize the surgical field with 70% ethanol and 10% iodide dermic solution. With a forceps part the mouse hair along a 2 cm length just above the joint of the hind leg. Shave the surgical area.
3. With a scalpel, make an incision 1.5 cm length of first, the cutaneous tissue, then with scissors cut the muscular tissue. Apply a slight pressure to both sides of the incision, which will force the kidney out of the abdominal cavity.
4. Apply saline with a cotton-bud to keep the kidney moist. If you have difficulties in exposing the organ, use a flat forceps to pull out the surrounding adipocyte tissue to which the kidney is attached. Maintain the kidney out of the retroperitoneal cavity by a cotton bud placed underneath the organ.
5. With two fine forceps, make a 2-3 mm hole in the capsule (avoid touching the parenchyma to prevent bleeding). During the whole surgical procedure keep the exposed kidney capsule continuously moistened.
6. Insert carefully the lobes under the kidney capsule, by maintaining the wall of the capsule opened and slide the graft under the capsule towards the edge pole. Place up to 6 lobes per kidney.
7. Replace the kidney in the retro-peritoneal cavity. Suture the muscle aponeuroses, then the skin with individual surgeon knots. Wet the wound with 10% iodide povidone solution. Place the mouse on a heated pad at 37 °C.
8. Survey the transplanted mouse until full recovery from anesthesia by control of cardiac, respiratory and whisker movements, mucosal colors until total recovery seen by self contained movements.
   NOTE: We recommend injection of analgesic solution (Buprenorphin, 0.1 mg/kg) intra muscle just after the surgery and the 2 days following surgery to prevent post-surgery pain.
9. Keep the operated animal in isolation until fully recovered. Inspect every other day the stitches of the wound to verify and prevent infection. Never observed wound infection following this procedure.
   NOTE: Weekly analysis of peripheral blood cells from the CD3^-/- grafted mice allow us to detect the thymic derived populations originated from either E13 or E18 progenitors using the CD45 allotypic marker and T cell lineage specific antibodies (Figure 3).

9. Analysis of the TSP progeny by Flow Cytometry

1. Tease the FTOCs individually to obtain single cell suspensions (see protocol section 4).
2. Stain cell suspensions from individual lobes with an antibody mixture containing antibodies recognizing CD45.1 PECy7, CD45.2 AmCyan, CD4 APCCy7, CD8 APC, CD3 Pacific Blue, Vγ5 FITC, Vδ1 PE and incubate as described in section 5.
3. Re-suspend the cells in HBSS + 1% FCS + propidium iodide and analyze in a flow cytometer (see results in Figure 2).