Work from the authors laboratory has been supported by USDA/CSREES/NRICGP (2007-35203-18213); NIH/NCRR (2 R01 RR17359-06), NIH/NIEHS (1 R21 ES014856-01A2) and Alberta Innovates &#x2013; Health Solutions.

PART A. Germ cell transplantation in mice
1. Preparation of recipient mice
<ol>
 <li>Recipients should be immunologically tolerant (either genetically matched to donors or immune-deficient) to the donor testis cells.</li>
 <li>Recipients should be either naturally devoid of spermatogenesis (e.g. W/ Wv mice) or depleted of endogenous germ cells. Germ cell depletion can be achieved by irradiation or chemotherapeutic drugs such as Busulfan. In this protocol, we describe the method of preparing recipient mice with Busulfan.</li>
 <li>Treat recipients at 4-6 week of age through i.p injection of Busulfan. The optimal dose of Busulfan is strain dependent. In commonly used recipient strains, a dose of 40-50 mg/kg is sufficient to deplete endogenous germ cells (e.g. 44 mg/kg for nude mice, 50 mg/kg for B6/129 F1 recipients).</li>
 <li>Dissolve Busulfan powder in DMSO and then add an equal volume of sterile distilled water to make a final concentration of 4 mg/ml. Keep solution warm at 35-40&deg;C before use to avoid precipitation of Busulfan. Discard solution if precipitation is observed.</li>
 <li>After Busulfan treatment, allow at least one month before using recipients. Recipients can be used between 1 month and 3 months post-Busulfan treatment.</li>
</ol>
2. Preparation of microinjection pipettes
<ol>
 <li>Choose borosilicate glass pipettes (capillary tubes) with a 1.0 mm outer diameter, a 0.75 mm inner diameter, and a length of 3 inches.</li>
 <li>Siliconize glass pipettes with Sigmacote, rinse pipettes with methanol and blow dry.</li>
 <li>Pull the pipettes using a pipette puller. Different pipette puller machines have different settings, therefore, test a few settings. Generally, a setting similar to that used for making enucleation pipettes may work.</li>
 <li>Break the pipette tips under dissecting microscope prior to use to achieve a diameter of approximately 50 &mu;m at the tip.</li>
</ol>
3. Preparation of donor cells for transplantation
<ol>
 <li>Choise of donor strain is dependent on the experimental question studied. If quantification of spermatogenesis is used as endpoint, use of donors with an easily identifiable genetic marker such as Lac-Z (e.g. B6.129S7-Gt(ROSA)26Sor/J from Jackson Laboratory) or GFP (e.g C57BL/6-Tg(CAG-EGFP)1Osb/J from Jackson Laboratory).</li>
 <li>Prepare 7 ml of collagenase in DMEM at 1 mg/ml, 4 ml of trypsin-EDTA (0.25% trypsin plus 1mM EDTA), and 2 ml of DNase in DMEM at 7 mg/ml. This amount is for digesting 2 donor mouse testes.</li>
 <li>Collect testes aseptically and remove the tunica albuginea. Spread seminiferous tubules gently with fine forceps to facilitate enzymatic digestion.</li>
 <li>Transfer tubules into collagenase, incubate at 37 &deg;C for 5-10 min, and agitate frequently.</li>
 <li>Wash tubules twice by spinning (200-300 g for 3 min) and re-suspending in PBS w/o Ca2++.</li>
 <li>Re-suspend tubules in trypsin-EDTA and shake until they become sticky and cloudy. Monitor the digestion of tubules at 37 &deg;C as it should occur within 1-2 min.</li>
 <li>Add DNase and shake well. Incubate for 1-2 min.</li>
 <li>Add 3 ml of FBS to stop the action of enzymes.</li>
 <li>Filter cell suspension using a nylon mesh with 40-70 &mu;m pore size to remove cell/tissue chunks.</li>
 <li>Collect cells at 600 g for 5 min and re-suspend cells in a small amount of DMEM (&lt;100 &mu;l).</li>
 <li>Count cells and adjust the volume to a desired final concentration (usually 100 x 106). Keep cell suspension on ice prior to use.</li>
 <li>Each Busulfan-treated mouse testis will be filled with only 10-15 &mu;l of cell suspension. Due to potential waste and leakage, aim to have 30-50 &mu;l per testis.</li>
</ol>
4. Transplantation procedure (Figure 1)
<ol>
 <li>Anaesthetize recipients following approved protocols.</li>
 <li>Place in dorsal recumbency and surgically prepare the abdominal area. Make a ~1cm midline abdominal incision to expose the abdominal wall. Lift abdominal wall by using small forceps at the point of the white line to avoid accidentally injuring abdominal organs, and proceed to make a ~0.5 cm incision at the midline of the abdominal wall to expose the peritoneal cavity.</li>
 <li>Use one iris forceps to hold the abdominal wall, and use another pair of iris forceps to search for the fat pads attached to the epididymis and testis in the peritoneal cavity. Gently pull the fat pad out until the testis is exteriorized and the testicular artery and epididymis are clearly visible. Work on one testis at a time.</li>
 <li>Place a thin sterile drape made from autoclaved index cards underneath the fat pad/testis for better visual identification (optional). The drape also works to absorb fluid.</li>
 <li>Add a drop of Trypan Blue dye into the cell suspension and carefully load the cell suspension into the polyethylene tubing connected to a 1 ml syringe. Attach the pulled pipette into the tubing and gently force the cell suspension into the pipette by applying pressure to the syringe.</li>
 <li>Identify the efferent ducts (that connect the testis to the epididymis) and gently remove fat tissue around the ducts. Work carefully as the ducts and the membrane around them are translucent.</li>
 <li>Carefully insert the pipette into a duct in the bundle of efferent ducts, gently thread a few mm toward the testis.</li>
 <li>While avoiding moving the injection pipette, reach for the syringe, gently depress the plunger of the syringe to ensure that the suspension flows into the rete testis and seminiferous tubules begin to fill.</li>
 <li>The injection rate and flow of cell suspension is regulated by thumb pressure. Avoid sudden increase in pressure; monitor the movement of suspension in tubules.</li>
 <li>Stop the injection when almost all surface tubules have been filled before the testis starts to become ischemic.</li>
 <li>Return the testis to the abdominal cavity. Repeat procedure on the contralateral testis. Suture the abdominal wall with 6-0 silk suture and close the skin with metal wound clips. Monitor mice on a warming pad until full recovery.</li>
</ol>
5. Analysis of the recipient testes
<ol>
 <li>Allow 2-4 months after transplantation before analysis.</li>
 <li>Euthanize the recipient mouse according to animal care and use guidelines and collect the testis and epididymis into PBS. When a Lac-Z transgenic donor strain has been used, the epididymis can be used as a positive staining control as it has endogenous beta-galactosidase activity.</li>
 <li>For detection of donor cells by Lac-Z staining, fix the testis for 1-2 hour at 4 &deg;C in 4% paraformaldehyde (PFA) in PBS. Rinse 3 x 30 min at room temperature in lacZ rinse buffer.</li>
 <li>Incubate overnight at 37 &deg;C in lacZ staining solution. Testis can be stained as a whole or after dispersing the tubules.</li>
 <li>Fix and store in 10% neutral buffered formalin. If sections are needed use neutral fast red as counter-stain.</li>
</ol>
PART B. Ectopic xenografting in mice
(From Dobrinski and Rathi 200815, and Rodriguez-Sosa et al. 201116).
1. Collection of donor tissue
<ol>
 <li>Obtain testis tissue by castration or biopsy from a donor male.</li>
 <li>Place testis in PBS or biopsies into culture medium, maintaining sterile conditions.</li>
 <li>Keep the collected tissue on ice and transport to the laboratory.</li>
</ol>
2. Preparation of donor tissue
<ol>
 <li>Perform in the tissue culture hood to maintain sterility.</li>
 <li>Wash each testis in ice-cold PBS containing antibiotics two to three times before transferring into a culture-dish with PBS. In the case of biopsies, wash testis fragments two to three times with ice-cold culture medium containing antibiotics by spinning at 150g for 2 min and resuspending in fresh ice-cold culture medium.</li>
 <li>For intact testes, remove tunica vaginalis by making an incision along the surface and extrude the testis. Remove from testis all annex structures (spermatic cord, epididymis, connective tissue). Wash testes once in cold PBS and transfer into a culture-dish with PBS.</li>
 <li>Carefully remove the tunica albuginea of the testis by using a scalpel blade and a pair of scissors. If the testis is very small, the tunica can be removed by squeezing the testicular tissue out of the tunica through a small incision made on one end while holding the tunica with a pair of small forceps on the other end.</li>
 <li>Depending on the size of the testis either the whole testis tissue can be cut into small pieces of around 1 - 2 mm3 in size using curved forceps and a scalpel blade, or large pieces of testis tissue can first be removed from the testis and then cut into smaller pieces. All this should be done in ice-cold culture medium and under sterile conditions in a small culture dish (60 x15 mm).</li>
 <li>Transfer the prepared tissue fragments to ice-cold culture medium in small culture-dishes on ice until grafting.</li>
</ol>
3. Castration of recipient mouse
<ol>
 <li>Anaesthetize mouse as above and prepare sterile surgical field by clipping the hair (not necessary in nude mice), wiping with 70% Ethanol and Betadine solution.</li>
 <li>Make a 0.5 -1 cm ventral midline skin incision to expose the abdominal wall.</li>
 <li>Carefully expose the testis, the testicular artery and epididymis as described in PartA, 4.2-4.3.</li>
 <li>Detach the tail of the epidydimis from the gubernaculum by blunt dissection.</li>
 <li>Ligate the testicular artery, and the vas deferens together with the blood vessel with silk, and section the ligated structures by cutting between the testis and the ligature.</li>
 <li>Repeat the procedure for the second testis.</li>
 <li>Suture the abdominal wall with one or two surgical stitches.</li>
 <li>Close the skin incision with one or two Michel clips.</li>
</ol>
4. Ectopic xenografting
<ol>
 <li>Position the mouse in ventral recumbency and prepare a sterile surgical field on its back as above.</li>
 <li>Depending on how many grafts are to be inserted (generally 4-8/mouse), make ~0.5 cm long skin incisions on each side of the mid line of the back of the mouse.</li>
 <li>Use forceps to hold a border of the skin incision, and make a subcutaneous cavity by teasing apart the connective tissue using scissors.</li>
 <li>Using an iris forceps place a piece of testis tissue deep into the subcutaneous cavity, holding the border of the skin incision with another iris forceps.</li>
 <li>Close the skin incision with one Michel clip and keep mouse on heating pad until it starts to recover from anaesthesia.</li>
 <li>Transfer the mouse to a cage with additional insulation and cover and monitor until mice are fully recovered.</li>
</ol>
5. Collection of testis xenografts for analysis and sperm harvesting
<ol>
 <li>Euthanize the host mouse according to animal care and use guidelines, and make a midline skin incision on the back skin running from the tail to the neck and open skin. This exposes the grafts which can be located either on the subcutaneous tissue or attached to the skin.</li>
 <li>Carefully remove the grafts using a pair forceps and a pair of scissors.</li>
 <li>Record number of grafts recovered, size and weight of individual grafts.</li>
 <li>Retrieve the seminal vesicles from the abdomen of the mouse and record their weight as an indication of testosterone production by the grafted tissue.</li>
 <li>For histological analysis
 <ol class="lalpha">
 <li>Suspend xenografts into sample vial containing Bouin's solution (or other fixative) in a volume ~10x that of the xenograft, and label vial appropriately.</li>
 <li>Incubate overnight in the refrigerator followed by washing at least 3 times in 70% ethanol at intervals of 24 hours preferably.</li>
 <li>Proceed for processing and embedding in paraffin.</li>
 </ol>
 </li>
 <li>For sperm harvesting
 <ol class="lalpha">
 <li>Wash xenografts by spinning them down at 300g for 1 min and resuspending them in culture medium containing antibiotics.</li>
 <li>Cut grafts into small pieces and mince carefully with the forceps in a tissue culture dish containing 3 - 5 ml of culture medium.</li>
 <li>Filter minced tissue through the 40 &mu;m cell strainer.</li>
 </ol>
 </li>
</ol>
PART C. Representative Results
1. Germ cell transplantation
If the transplanted donor cell suspension contains spermatogonial stem cells carrying a genetic marker such as the LacZ transgene, colonization of donor SSCs in the recipient testis can be visualized by X-gal staining as distinctive blue segments of the seminiferous tubules after 2-3 month post-transplantation3 (Figure 2A). A well- established colony should have a long dark blue stretch of completely filled segments with two or more layers of blue cells closer to the center, and relatively weaker stained regions at both ends where a network of single, paired or small groups of blue cells is apparent3 (Figure 2B). A cross section of the dark blue seminiferous tubule region should reveal well-established and well-organized spermatogenesis with blue germ cells at various differentiation stages (Figure 2C).
2. Testis tissue xenografting
The viability of testis tissue after transplantation is inversely correlated with the developmental stage of the donor; the best outcome is obtained when tissue from newborn males is used, while adult tissue shows a high tendency to degenerate and die17-21. Generally, xenograft success decreases as donor tissue undergoes meiosis of the first wave of spermatogenesis. Time to full development of immature donor tissue and complete spermatogenesis is species specific, and often somewhat shorter in comparison with testes in situ. The number of seminiferous tubules with complete spermatogenesis is variable according to the species. While in sheep, goats and pigs that number is greater than 50%, in cattle and cats it is less than 15% 14(Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/3545/3545fig1.jpg" /> 
Figure 1. Germ cell transplantation procedure. Place recipients in dorsal recumbency after anaesthesia and make a ~0.4-inch midline abdominal incision (A). Expose testis by withdrawing the fat pad attached to the epididymis and testis and place a thin sterile drape underneath the fat pad/testis for better visual identification (B). Position the testis and epididymis so that the efferent ducts buried in the fat pad are discernible (C, D, E). Identity the efferent ducts and gently remove fat tissue around the ducts. A piece of colored paper or plastic can be placed underneath the ducts for better visualization (G). Break or grind the pipette tip according to the size of the ducts and load cell suspension into the pipette (H). Carefully insert the pipette into a duct in the bundle of efferent ducts, gently thread a few mm toward the testis (the arrow in H shows the direction of pipette injection and threading). A testis with successful injection into the seminiferous tubules is shown (I). Ts: Testis; Ep: epididymis.
<img alt="Figure 2" src="/files/ftp_upload/3545/3545fig2.jpg" /> 
Figure 2. Representative results for germ cell transplantation A) An injected testis stained with X-gal. The blue segments of the seminiferous tubules represent established colonies from transgenic donor SSCs (LacZ transgene). B) Higher magnification of a SSC colony at 3 month post-transplantation. The dark blue region in the center represents complete spermatogenesis and the pale blue regions at the ends represent growing extension of the colony. C) A histological section of the dark blue seminiferous tubule (3 month post-transplantation) shows well organized spermatogenesis. Scale bars in B and C are 200 &mu;m and 30 &mu;m, respectively. (Figures adapted from Annu Rev Cell Dev Biol. 2008;24:263-86 and Biol Reprod. 1999 Jun;60(6):1429-36).
<img alt="Figure 3" src="/files/ftp_upload/3545/3545fig3.jpg" /> 
Figure 3. Ectopic xenografting of immature testis tissue from large animals into immunodeficient mice. Fragments of immature donor testis (~1 mm3) transplanted under the dorsal skin of immunodeficient mice (A) are able to survive and respond to mouse gonadotropins. As a result, testis tissue undergoes complete development, including formation of fertilization competent sperm (B). Once testis xenografts are collected (C) they can be used for analysis or to obtain sperm (D) for ICSI (E) and embryo production (F). Bars equal 50 &mu;m (B, E and F) or 10 &mu;m (D). (Modified from Rodriguez-Sosa and Dobrinksi 200914).

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+and+cryopreservation+of+immature+non-human+primate+testicular+tissue+on+spermatogonial+stem+cell+potential+in+xenografts.">Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.</a> Human Reproduction. 22 (4), 1060-1060 (2007).</li><li>Rathi, R., Zeng, W., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+testicular+tissue+from+infant+monkeys+after+xenografting+into+mice.">Maturation of testicular tissue from infant monkeys after xenografting into mice.</a> Endocrinology. 149 (10), 5288-5288 (2008).</li><li>Honaramooz, A., Li, M. W., Penedo, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+maturation+of+primate+testis+by+xenografting+into+mice.">Accelerated maturation of primate testis by xenografting into mice.</a> Biology of Reproduction. 70 (5), 1500-1500 (2004).</li></ol>

No conflicts of interest declared.

1. Germ cell transplantation
Germ cell transplantation provides the only functional assay for unequivocal confirmation of the presence of spermatogonial stem cells (SSCs) in a cell population. Only SSCs can home to and colonize the SSC niche at the basement membrane and initiate donor-derived spermatogenesis. Germ cell transplantation made it possible to study and manipulate SSCs in an unprecedented manner. The technique has been used to produce transgenic animals through genetic manipulation of...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Collagenase (type IV)</td>
 <td>Sigma</td>
 <td>C5138</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Invitrogen</td>
 <td>25200-056</td>
 </tr>
 <tr>
 <td>DNaseI</td>
 <td>Sigma</td>
 <td>DN25</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 </tr>
 <tr>
 <td>Trypan blue stain</td>
 <td>Invitrogen</td>
 <td>15250-061 </td>
 </tr>
 <tr>
 <td>Nylon mesh cell strainer</td>
 <td>BD biosciences</td>
 <td>352340 (40&mu;m) 
 352350 (70&mu;m)</td>
 </tr>
 <tr>
 <td>Busulfan</td>
 <td>Sigma</td>
 <td>B2635</td>
 </tr>
 <tr>
 <td>Thin-Wall Glass Capillaries</td>
 <td>World Precision Instrument</td>
 <td>TW 100-3</td>
 </tr>
 <tr>
 <td>BD intramedic plyethylene tubing (PE100) </td>
 <td>BD </td>
 <td>CA-63018-725</td>
 </tr>
 <tr>
 <td>Ethicon 6-0 Silk Suture</td>
 <td>Ethicon</td>
 <td>706G</td>
 </tr>
 <tr>
 <td>Wound clips</td>
 <td>BD</td>
 <td>427631</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma</td>
 <td>SL2</td>
 </tr>
 <tr>
 <td>X-gal</td>
 <td>Sigma</td>
 <td>B4252</td>
 </tr>
 <tr>
 <td>Potassium Ferrocyanide</td>
 <td>Sigma</td>
 <td>P9387</td>
 </tr>
 <tr>
 <td>Potassium Ferricyanide</td>
 <td>Sigma</td>
 <td>P3667</td>
 </tr>
 <tr>
 <td>magnesium chloride</td>
 <td>Sigma</td>
 <td>208337</td>
 </tr>
 <tr>
 <td>sodium deoxycholate</td>
 <td>Sigma</td>
 <td>D6750</td>
 </tr>
 <tr>
 <td>N,N-Dimethylformamide</td>
 <td>Sigma</td>
 <td>D4551</td>
 </tr>
 <tr>
 <td>Igepal CA-630</td>
 <td>Sigma</td>
 <td>18896</td>
 </tr>
 </tbody>
</table>

germ cell transplantation and testis tissue xenografting in mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

germ-cell-transplantation-and-testis-tissue-xenografting-in-mice

Research

JoVE Journal

Biology

29.6K Views.  University of Calgary. Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Video: Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...