Renal Subcapsular Transplantation of 2′-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice
Summary
We provide a simple and efficient method to transplant 2’-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.
Abstract
The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2’-deoxyguanosine (to deplete donor’s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient’s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.
Introduction
The thymus is the central immune organ, within the thymus thymocytes undergo positive and negative selection, and become mature T cells1,2. Abnormal positive or negative selection results in immunodeficiency or autoimmune pathologies respectively3,4. Therefore, thymus organ transplantation is an important approach to study the process of T cells selection in the donor’s thymus. This method is particularly crucial when analyzing thymic epithelial function mediated by gene mutations which cause embryonic lethal phenotype when mutated5.
In order to study the maturation of a recipient’s T cells in the transplanted thymus, depletion of donor’s lymphocytes within the thymus is necessary. For this purpose, embryonic 14-, 15- or 16-day (E14, E15, E16) thymus is usually selected6,7. Thymus from more mature stages can also be successfully depleted of the donor’s lymphocytes by treating with 2’-deoxyguanosine. However, a detailed protocol for depleting lymphocytes and use of older thymus culture has not previously been described8,9. While transplantation protocols have been introduced by several studies10,11, further modification and improvement of these protocols is necessary.
Our protocol is separated into two parts: (i) Depletion of T lymphocytes from late developmental stage E18.5 thymus by culture in 2’-deoxyguanosine-containing media. (ii) Transplantation of the cultured thymus into recipients. In this procedure, we developed a simple way to deliver the large tissue (E18.5 thymus) into the renal capsule with reduced chance of kidney injury. While focussing on later stage thymus, our protocol can also be used directly or with modifications for transplantation of thymus at various developmental stages or other similar sized tissues.
Protocol
The presented protocol adheres to the guidelines of the ethics committee of Jinan University regarding animal care.
NOTE: Materials used are listed in the Table of Materials.
1. Isolation of embryonic thymus
- Autoclave all surgical instruments before the experiment, and sterilize the bench/hood with 70% ethanol.
- Using carbon dioxide, anesthetize and euthanize the pregnant female mouse (18.5 days post successful mating). Then wipe the abdominal region with 70% ethanol.
NOTE: Here we mated Insm1+/lacZ females and Insm1+/lacZ males. Intraplacental injection of pentobarbital were performed before isolation of embryos from the uterus and decapitation were performed for each embryo. - Using scissors, make a "V" shaped cut on the abdomen starting from the bladder and running until each horn of the uterus.
- Using the scissors, cut the mesometrium and cervix/vagina, and collect the uterus. Place the uterus in a Petri dish containing cold phosphate-buffered saline (PBS) on ice. Next, expose the embryos which are in the enveloped decidua, by cutting the anterior uterine wall from one uterine horn to the other. Using fine tweezers, peel back the enveloped decidua tissues and cut the umbilical cord to release the embryos. Place all the embryos in a new Petri dish on ice until the isolation of the thymus.
- Wipe an embryo with 70% alcohol and place it in a new Petri dish. From this step on, ensure that sterile conditions are maintained.
- By cutting close to the lower jaw with scissors, remove the head of the embryo and drain the blood with a paper towel. Next, fix the embryo on the same Petri dish in supine position.
NOTE: We cut a piece of the tail of each embryo for Insm1 and lacZ genotyping. - Using scissors, cut the lateral chest wall horizontally along the axillary front, and then cut the diaphragm to open the chest. The thymus should now be visible as two white lobes located in front of the trachea and adjacent to the heart.
- Place bent-tip forceps behind the thymus and then pull off the thymus gently. Be sure to check the integrity of the thymus to confirm that it contains two jointed lobes.
- Wash the thymus with 1x PBS and trim off the connective tissues and blood vessels under a stereomicroscope.
2. Culture of the isolated embryonic thymus
- Add 500 µL of culture medium (RPMI1640 + 15% fetal bovine serum (FBS) + 100 U/mL penicillin and 100 µg/mL streptomycin) to each well of a 24-well plate. Transfer the clean thymi into the wells with one thymus per well. Figure 1 displays the isolated thymus in culture media.
- To each thymus-containing well add 2'-deoxygranosine to a final concentration of 1.25 mM.
- Culture the isolated thymus for eight days, refreshing both the culture media and 2'-deoxygranosine every two days.
3. Establish the subcapsular space in the renal capsule
- To prepare the needle and the clipped infusion tube (Figure 2), cut the scalp vein needle on the tube part at a 45° angle using scissors.
- Weigh the nude mouse and then anesthetize it with a pentobarbital (1.5%) injection (75 µg/g body weight).
- When no reflex following toe pinch is observed, place the mouse on the operating table in a right lateral position.
- With a 0.5% povidone iodine swab, disinfect the skin twice in the surgical area from the inside to the outside of the body.
- Using scissors, make a 5–9 mm skin incision parallel to the spine in the left renal area (between the last rib and the iliac crest). Next, open the abdominal cavity by cutting through the subcutaneous tissue and muscle and expose the kidney.
- With the kidney exposed, use a pair of tweezers in one hand to lift the muscle and fat tissue off the spine-side incision edge. With the other hand, gently squeeze the kidney out (alternatively, the kidney can be squeezed out using fingers of both hands).
- To ensure that the renal capsule is moist during the surgery, wet the surface of the kidney with saline (0.9% NaCl).
- Create a nick in the kidney capsule, and gently scratch the renal capsule on the lower right side using the needle tip prepared in step 3.1. The size of the nick should be 1/2–2/3 widths of the kidney; do not scratch on the kidney.
- Slide the infusion tube prepared in step 3.1 into the nick on the renal capsule. Gently dissociate the renal capsule with the kidney along the kidney's long side until reaching 3–4mm inside the kidney capsule. Draw back the infusion tube; the kidney subcapsular space is established.
4. Transplant the embryonic murine thymus
- Wash the thymus cultured in step 2.3 twice in saline to deplete the culture media.
- Connect the clipped infusion tube prepared in step 3.1 to a syringe at its syringe-connecting interface. Aspirate the prepared thymus into the infusion tube slowly.
- Gently insert the clipped infusion tube into the renal capsule and reach the superior pole. Deliver the thymus into the renal capsule; retract the tube slowly while simultaneously pushing the plunger of the syringe gently.
- Using an alcohol lamp, slightly heat the needle prepared in step 3.1. After ensuring that the whole thymus is inside the subcapsular space, use the heated needle to cauterize the nick.
- After cauterization, restore the kidney in the abdominal cavity. Suture the peritoneum and muscle.
- Using a modified interrupted vertical mattress suture, close the skin incision (tie at least three knots and cut away any excess thread).
- Using a povidone iodine swab, disinfect the incision. To relieve pain, subcutaneous injection of flunixin (2 µg/g of body weight) was performed during the surgery and then for 3 days after the surgery.
- Until fully recovered from anesthesia, keep the mouse warm under the infrared lamp.
- Keep the thymus in the recipient’s kidney capsule for 8 weeks before dissecting the transplanted thymus and conducting phenotypic analysis as previously described5,8,9.
Representative Results
Here we show the isolated E18.5 thymus containing two complete lobes (Figure 1). Additionally, we show the scalp vein needle that was clipped to form a bevel on the infusion tube (Figure 2). Next, we also show a representative image of the position of the thymus that was transplanted in the renal capsule (Figure 3A) and the thymus after 8 weeks of growth within the recipient mice (Figure 3B). To determine whether the T cells were produced in nude mice transplanted with a thymus, in both the transplanted thymus and the peripheral blood, we detected the cell populations using CD4 and CD8 antibody staining and flow cytometry analysis. The peripheral blood was collected from the retro-orbital sinus as previously described12. We found the T cells were produced in both the transplanted thymus and the peripheral blood of nude mice transplanted with a thymus. However, no T cells were detected in peripheral blood of non-transplanted nude mice (Figure 4). To determine the source of T cells, we checked Insm1 and lacZ genes in the peripheral white blood cells using genotyping methods routinely used in our laboratory and described previously13,14. Since the donor embryo Insm1 gene was replaced by the lacZ gene in one or both alleles, when the T cells were co-transplanted with thymus from donor, we could detect the lacZ gene in the genome of the T cells collected from peripheral blood of recipient, which indicates that they were produced by the donor thymus. Additionally, as no lacZ gene was present, lacZ would not be detected when the T cells were generated from recipient’s hematopoietic cells. We did not detect lacZ gene in the peripheral T cells indicating that the T cells were generated from recipient (Figure 5).
Figure 1: Thymus isolated from E18.5 embryos. Please click here to view a larger version of this figure.
Figure 2: Thymus delivering tools made from the scalp vein needle. The scalp vein needle was cut at the infusion tube part close to the needle at an angle of 45° to create a bevel. Both the needle and the infusion tube were used in the procedure. Please click here to view a larger version of this figure.
Figure 3: Thymus transplanted into the renal capsule. (A) Freshly transplanted E18.5 thymus in the renal capsule. (B) Thymus in the renal capsule after 8-week growth in recipient. Please click here to view a larger version of this figure.
Figure 4: Flow cytometry analysis of the T cells isolated from the transplanted thymus, blood of thymus-transplanted and non-transplanted nude mice. CD4 and CD8α antibodies were used for T cells staining. CD4+CD8+ double positive cells, CD4+ single positive, CD8+ single positive and CD4–CD8– double negative cells are shown in each of the quadrants as indicated. Please click here to view a larger version of this figure.
Figure 5: Identifying the source of the peripheral blood T cells in thymus transplanted nude mice. Genotyping of lacZ gene and Insm1 gene in peripheral blood white cells is shown. Ladder: DNA marker, + : positive control DNA, -: negative control DNA, Anim1: DNA from peripheral white blood cells of nude mouse transplanted with Insm1lacZ/lacZ thymus, Anim2: DNA from peripheral white blood cells of nude mouse transplanted with Insm1+/lacZ thymus. Please click here to view a larger version of this figure.
Discussion
Renal subcapsular transplantation of embryonic thymus is an important method to study the thymic epithelial cells function and the process of T cells maturation in vivo. Although there are several experimental studies on embryonic thymus organ culture and transplantation6,7, our protocol provides a simple alternative procedure on murine embryonic thymus culture and renal subcapsular transplantation for older thymus tissue.
Our protocol improves upon previous protocols by incorporating several different modifications 6,7,10,11. First, instead of E14-E16 thymus, we utilized the thymus isolated from E18.5 for transplantation. The advantage is that thymus at this later developmental stage contains relatively mature thymic structures and epithelial cell populations. Although the newborn or adult mice are an alternative source of mature thymus, if perinatal lethal phenotype occurs as a result of gene manipulation, such as mutations in Jmjd6 or Insm1 genes5,13, this method provides a viable alternative for the study of mature thymus. A second modification is the prevision of a culture method of E18 thymus before transplantation. Additionally, a third modification occurs in the transplantation procedure, in which we used the needle tip to create a nick on the renal capsule instead of picking and cutting the renal capsule with tweezers and scissors. This modification reduced both renal capsule damage and injury of the kidney. One final modification is in the suture step. The modified interrupted vertical mattress suture eliminates the outside suture line on the skin and therefore prevents the opening of the incision due to biting.
While this protocol is used for E18.5 thymus transplantation into the kidney capsule, it can be modified for transplantation of the thymus at other developmental stages or for other tissues with similar sizes. Additionally, the materials used can be modified accordingly by different users from different areas, especially with respect to anesthesia reagents which may be restricted by local laws. The dosage of pentobarbital used in our protocol is 75 µg/g body weight. However, the maximal dosage should be no more than 100 µg/g body weight to prevent death of the anesthetized animals. Although the transplantation of the thymus into the renal capsule is an efficient method for the functional study of the thymus in vivo, some limitations exist in the method presented above. These limitations include risk of the thymus dropping out of the kidney capsule during the 8 weeks in vivo growth period (1 in 12). Secondly another limitation is the death of mice after the surgery (6 in 30). However, this death is mainly caused by the overdose of the pentobarbital. As such, other allowed methods of anesthesia can be employed.
In summary, we provide a simple and efficient protocol to isolate and culture the E18.5 thymus and to then subsequently transplant the thymus into the renal capsule. This then allows for the analysis of the thymic epithelial cells function and the process of T cells maturation.
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Start Package of Jinan University to S.J. and by Science and Technology Program of Guangzhou China (Grant No. 201704020209 to S.J.). We thank Amy Botta (Department of Biology, York University, Toronto, ON M3J 1P3, Canada) for proofreading and editing of the manuscript.
Materials
| 0.5% Povidone iodine | Shanghai Likang Distinfectant Hi-Tech Co.Ltd | 20171113 | |
| 0.9% Sodium Chloride Injection | Shandong Qilu Pharmaceuyical Co.Ltd | 2C17112101 | |
| 1 mL Sterile syringe | Solarbio | YA1090 | |
| 2’-Deoxyguanosine | MEC | HY-17563 | 1M in DMSO, 1:800 using (final 1.25mM) |
| 24 Well Plate | Corning Incorporated | Costar 3524 | |
| 4-0 Surgical suture needles with thread | NingBo Cheng-He Microsurgical Instruments Factory China | YY0166-2002 | |
| 60mm Cell Culture Dish | Corning Incorporated | 430166 | |
| 70% ETOH | LIRCON | 20181221 | |
| APC anti-mouse CD8a antibodies | Biolegend | 100711 | 1:100 |
| Bent-tip fine forceps, JZ 10 cm | Shanghai Medical Devices Group Co.,Ltd. | JD1060 | To sterilize before use |
| Cefmetazole Sodium for Injection | Sichuan Hexin Pharmaceutical co,Ltd | 17062111 079 | 6mg in 0.5ml 0.9% NaCl solution, 7.5ul/g body weight |
| Dissecting scissors, JZ 10 cm | Shanghai Medical Devices Group Co.,Ltd. | JC2303 | To sterilize before use |
| Fetal bovine serum (FBS） | GIBCO | 10270-106 | |
| Fine forceps, JZ 10 cm | Shanghai Medical Devices Group Co.,Ltd. | JD1050 | To sterilize before use |
| Flow cytometry | BD | FACSCanto II | |
| Flunixin meglumine | MACLIN | F810147 | 1mg in 1ml 0.9% NaCl solution,2ul/g body weight |
| Forceps, Dumont#5 | World Precision Instruments | 14098 | To sterilize before use |
| Infrared lamp | OTLAN | MT-810 | |
| Needle holder, JZ 14 cm | Shanghai Medical Devices Group Co.,Ltd. | J32010 | To sterilize before use |
| PE anti-mouse CD4 | Biolegend | 100511 | 1:100 |
| Penicillin-Streptomycin mixture | GIBCO | 15140122 | 1:100 |
| Pentobarbital sodium salt | Sigma | P3761 | 1.5% solution in PBS, 75ug/g body weight |
| RPMI1640 Medium | GIBCO | C14-11875-093 | |
| Scalp vein needle | Shanghai Kindly Medical Instruments Co., Ltd | XC001 | |
| Spring scissors | VANNAS | S11014-12 | To sterilize before use |
| stereomicroscope | OLYMPUS | SZ61 | |
| Sterile 15cm cotton swab | Guangzhou Haozheng | 20150014 | |
| Sterile gauze 5 cm x 7 cm-8P | Guangzhou Haozheng | 20172640868 | |
| Sterile PBS (1x) | GENOM | GNM20012 | |
| Tissue forceps, JZ 12.5 cm | Shanghai Medical Devices Group Co.,Ltd. | J41010 | To sterilize before use |
References
- Klein, L., Kyewski, B., Allen, P. M., Hogquist, K. A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nature Reviews Immunology. 14, 377-391 (2014).
- Hogquist, K. A., Baldwin, T. A., Jameson, S. C. Central tolerance: learning self-control in the thymus. Nature Reviews Immunology. 5, 772-782 (2005).
- Spits, H., Touraine, J. L., Yssel, H., de Vries, J. E., Roncarolo, M. G. Presence of host-reactive and MHC-restricted T cells in a transplanted severe combined immunodeficient (SCID) patient suggest positive selection and absence of clonal deletion. Immunological Reviews. 116, 101-116 (1990).
- Nagamine, K., et al. Positional cloning of the APECED gene. Nature Genetics. 17, 393-398 (1997).
- Yanagihara, T., et al. Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus. Nature Communications. 6 (8820), (2015).
- Jenkinson, W., Jenkinson, E., Anderson, G. Preparation of 2-dGuo-Treated Thymus Organ Cultures. Journal of Visualized Experiments. (18), (2008).
- Bosselut, R., Vacchio, M. S. . T-Cell Development: Methods and Protocols. , (2016).
- Anderson, M. S., et al. Projection of an immunological self shadow within the thymus by the aire protein. Science. 298 (5597), 1395-1401 (2002).
- Takaba, H., et al. Fezf2 Orchestrates a Thymic Program of Self-Antigen Expression for Immune Tolerance. Cell. 163 (4), 975-987 (2015).
- Morillon, Y. M. 2. n. d., Manzoor, F., Wang, B., Tisch, R. Isolation and transplantation of different aged murine thymic grafts. Journal of Visualized Experiments. (99), e52709 (2015).
- Caetano, S. S., Teixeira, T., Tadokoro, C. E. Intravital imaging of the mouse thymus using 2-photon Microscopy. Journal of Visualized Experiments. (59), e3504 (2012).
- JoVE Science Education Database. Blood Withdrawal I. Lab Animal Research. , (2019).
- Gierl, M. S., Karoulias, N., Wende, H., Strehle, M., Birchmeier, C. The zinc-finger factor Insm1 (IA-1) is essential for the development of pancreatic beta cells and intestinal endocrine cells. Genes & Development. 20 (17), 2465-2478 (2006).
- Tao, W., et al. Haploinsufficiency of Insm1 Impairs Postnatal Baseline β-Cell Mass. Diabetes. 67 (12), 2615-2625 (2018).
