Supported by the Pasteur Institute, INSERM, Agence Nationale de Recherche ANR (Grant &#8216;Lymphopoiesis&#8217;), the REVIVE Future Investment Program and &#8220;La Ligue contre le Cancer&#8221;.

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Dissection of the Embryos Under a Horizontal Laminar Flow Hood
NOTE: Two days before the grafting experiment.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Place the uterus in a 90 x 15 mm Petri dish containing 40-50 ml DPBS and cut transversally between each embryo&#8217;s decidua.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Isolation of the Thymus
<ol>
	<li>Under the binocular magnifying lens, place the embryo, lying in supine position (ventral view), on a wet gauze bedding.</li>
	<li>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.</li>
	<li>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 &#8540;&#34; needles, without damaging the capsule.</li>
	<li>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.</li>
</ol>
4. Cell Suspensions
<ol>
	<li>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.</li>
</ol>
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).
<ol>
	<li>Incubate the thymocytes (E13 or E18) for 15-30 min at 4 &#176;C with a 100 &#956;l 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).</li>
	<li>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.</li>
	<li>Incubate the E18 biotin-labeled cells with streptavidin microbeads during 15 min at 4 &#176;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.</li>
	<li>Pass the cells on the LS column, according to the manufacturer&#8217;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.</li>
	<li>Re-suspend the pellet of either depleted E18 or total E13 thymocytes in 50 &#956;l 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 &#176;C in the dark.</li>
	<li>Wash the cells with 4 ml HBSS + 1% FCS and re-suspend the cells in 1 ml HBSS + 1% FCS with propidium iodide.</li>
	<li>Sort the TSPs Lin-CD44+CD117+CD24lowCD127+CD135+ cells in a FACS (with 4 lasers) onto a 1.5 ml tubes containing 200 &#956;l 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.</li>
</ol>
6. Colonization of E14 Thymic Lobes with Progenitors: Hanging Drop Technique
<ol>
	<li>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.</li>
	<li>Centrifuge sorted TSPs and re-suspend the cells in culture medium (OPTI-MEM complete medium with 10% FCS, penicillin (50 units/ml), streptomycin (50 &#181;g/ml) and 2&#946;-mercaptoethanol (50 &#181;M)) such that 35 &#956;l contain 500 TSP.</li>
	<li>Place a 35 &#956;l drop of cell suspension every other well of a 60 well Terasaki plate. 
	NOTE: 35 &#956;l drops can fuse if they are placed in contiguous wells.</li>
	<li>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.</li>
	<li>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 &#176;C + 5% CO2. After colonization, either culture E14 thymi in FTOC7, or graft under the kidney capsule of a recipient adult mouse 8.</li>
</ol>
7. Fetal Thymic Organ Culture
<ol>
	<li>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).</li>
	<li>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 &#176;C + 5% CO2.</li>
</ol>
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.
<ol>
	<li>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 &#956;l 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
9. Analysis of the TSP progeny by Flow Cytometry
<ol>
	<li>Tease the FTOCs individually to obtain single cell suspensions (see protocol section 4).</li>
	<li>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, V&#947;5 FITC, V&#948;1 PE and incubate as described in section&#160;5.</li>
	<li>Re-suspend the cells in HBSS + 1% FCS + propidium iodide and analyze in a flow cytometer (see results in Figure 2).</li>
</ol>

<ol><li>Rodewald, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thymus+organogenesis%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">Thymus organogenesis.</a> Annu Rev Immunol. 26, 355-388 (2008).</li>
<li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Journey+through+the+thymus%3A+stromal+guides+for+T-cell+development+and+selection%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat Rev Immunol. 6, 127-135 (2006).</li>
<li>Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MHC+class+II-positive+epithelium+and+mesenchyme+cells+are+both+required+for+T-cell+development+in+the+thymus%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.</a> Nature. 362, 70-73 (1993).</li>
<li>Douagi, I., Andre, I., Ferraz, J. C., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+T+cell+precursor+activity+in+the+murine+fetal+thymus%3A+evidence+for+an+input+of+T+cell+precursors+between+days+12+and+14+of+gestation%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation.</a> Eur J Immunol. 30, 2201-2210 (2000).</li>
<li>Calderon, L., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synergistic%2C+context-dependent%2C+and+hierarchical+functions+of+epithelial+components+in+thymic+microenvironments%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Synergistic, context-dependent, and hierarchical functions of epithelial components in thymic microenvironments.</a> Cell. 149, 159-172 (2012).</li>
<li>Adolfsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Flt3%2B+lympho-myeloid+stem+cells+lacking+erythro-megakaryocytic+potential+a+revised+road+map+for+adult+blood+lineage+commitment%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.</a> Cell. 121, 295-306 (2005).</li>
<li>Kondo, M., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91, 661-672 (1997).</li>
<li>Bhandoola, A., von Boehmer, H., Petrie, H. T., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Commitment+and+developmental+potential+of+extrathymic+and+intrathymic+T+cell+precursors%3A+plenty+to+choose+from%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from.</a> Immunity. 26, 678-689 (2007).</li>
<li>Ramond, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+waves+of+distinct+hematopoietic+progenitor+cells+colonize+the+fetal+thymus%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus.</a> Nat Immunol. 15, 27-35 (2014).</li>
<li>Allison, J. P., Havran, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunobiology+of+T+cells+with+invariant+gamma+delta+antigen+receptors%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">The immunobiology of T cells with invariant gamma delta antigen receptors.</a> Annu Rev Immunol. 9, 679-705 (1991).</li>
<li>Anderson, G., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fetal+thymus+organ+culture%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Fetal thymus organ culture.</a> CSH Protoc. , (2007).</li>
<li>Schmitt, T. M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+T+cell+development+from+hematopoietic+progenitor+cells+by+delta-like-1+in+vitro%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.</a> Immunity. 17, 749-756 (2002).</li>
<li>Barbee, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skint-1+is+a+highly+specific%2C+unique+selecting+component+for+epidermal+T+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Skint-1 is a highly specific, unique selecting component for epidermal T cells.</a> Proc Natl Acad Sci U S A. 108, 3330-3335 (2011).</li>
<li>Douagi, I., Colucci, F., Di Santo, J. P., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+earliest+prethymic+bipotent+T%2FNK+progenitor+in+murine+fetal+liver%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Identification of the earliest prethymic bipotent T/NK progenitor in murine fetal liver.</a> Blood. 99, 463-471 (2002).</li>
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The authors declare that they have no competing financial interests.

Two main assays can be used to analyze T cell differentiation ex vivo. The most recently reported is the co-culture of hematopoietic progenitors with BM stromal cells, OP9, expressing the ligands of Noth1, delta like 1 or 412. This 2-D assay is easy to perform, highly efficient and sensitive, allowing analysis at the single cell level. However, it neither supports T cell development beyond the stage of DP nor the generation of &#947;&#948; DETC13, both of which require direct interactions w...

T lymphocytes, bearing the &#945;&#946; or the &#947;&#948; T cell receptor (TcR), differentiate in a specialized organ, the thymus. The fully developed thymus is organized into two distinct regions: the cortex, where thymic progenitors develop and where thymocytes that productively rearrange the TcR &#946; and &#945; chain genes are rescued from programmed death (a process known as positive selection); and the medulla, where selected thymocytes with too strong reactivity to self-ligands are deleted (negative selection)1,2. The thymus originates from the endodermal layer of the third pharyngeal pouch that is later surrounded by mesenchymal cells3. It is colonized by hematopoietic progenitors starting at embryonic day E12 and, thereafter, continuous recruitment is required for normal T cell development4. Thymic immigrants evolve through successive developmental stages, orchestrated by a tightly regulated program, initiated and maintained by the activation of the Notch signaling pathway on thymocytes upon interaction with its ligand, delta like 4, expressed on thymic epithelial cells (TECs)5.
Thymocyte development starts at the so-called CD4-CD8- double negative (DN) stages. DN thymocytes can be further subdivided according to the expression of CD25 and CD44 into DN1 (CD25-CD44+), DN2 (CD25+CD44+), DN3 (CD25+CD44-) and DN4 (CD25-CD44-). CD24 (HSA) and CD117 (c-Kit) further subdivides the DN1 compartment into 5 subsets where DN1a and b correspond to early thymic progenitors (ETP). Thymocytes rearrange the TcR &#948;, &#946; and &#945; chains at the DN stage and undergo pre-TcR selection (DN3-DN4 stages). They further transit to the CD4+CD8+ double positive (DP) compartment where the TcR &#945; chain rearranges prior to positive and negative selection. At this stage most thymocytes are eliminated and only a small percentage (3-5%) reach the CD4+ or CD8+ mature T cell compartment.
The lymphoid differentiation pathway progresses through the stages of HSCs that generate multipotent progenitors (MPP) and lymphoid-primed multipotent progenitors (LMPP) that lost the erythrocyte and megakaryocyte potential6. LMPP are phenotypically defined by the absence of differentiated blood cell markers (lineage negative, Lin-), the expression of c-Kit (CD117), Sca-1 and Flt3/Flk2 (CD135) and the absence of detectable levels of the interleukin (IL)-7 receptor &#945; chain (IL-7r&#945; or CD127). LMPPs further differentiate into common lymphoid progenitors (CLP)7 that by that stage have lost the capacity to generate myeloid cells. CLP retain lymphocyte (B and T cell), NK cell, DC and innate lymphoid cell (ILC) potential, and differ from LMPP by the expression of CD127 and the absence of high levels of Sca-1.
Although the nature of the thymic settling progenitors (TSP) has been extensively debated8, it became recently clear that TSP change phenotype, differentiation potential and function, throughout development9. We performed in vitro and in vivo assays to characterize the TSP, isolated by FACS cell sorting from either E13 (first wave) or E18 (second wave). Fetal thymic organ cultures (FTOC) with irradiated thymic lobes colonized by equal numbers of E13 and E18 progenitors, bearing different allotypic markers, allowed following their progeny in a similar developmental environment and revealed cell intrinsic properties, different between both types of progenitors. Thymic lobes colonized by either E13 or E18 TSP allowed development without selection due to competition between both progenitors. In vivo transplantation of the colonized thymic lobes further showed that also the mature progeny of E13 and E18 TSP have different biologic properties in vivo. TSPs from the first wave rapidly generate T cells but give rise to low numbers of &#945;&#946; and &#947;&#948; T cells. Among the latter we detected V&#947;5V&#948;1 dendritic epithelial T cells (DETC), that have an invariant TcR, migrate to the epidermis where they exert a function in wound healing and are only produced during embryonic development10. In contrast, TSP from the second wave take longer time to generate high numbers of TcR+ T cells and are unable to generate DETC.

<table><tbody><tr><td>90 x 15 mm and 35 x 15 mm plastic tissue culture Petri dishes.</td><td>TPP</td><td>T93100/T9340</td><td>Sampling</td></tr><tr><td>26 G &amp;frac38;&quot; 0.45 x 10 mm syringes with needles</td><td>BD Plastipak</td><td>300015</td><td>Cell suspension</td></tr><tr><td>Nylon mesh bolting cloth sterilized 50/50 mm pieces.</td><td>SEFAR NITEX</td><td>03-100/32</td><td>Filtration of cells</td></tr><tr><td>Ethanol 70%.</td><td>VWR</td><td>83801.36</td><td>Sterility actions</td></tr><tr><td>Iodide Povidone 10% (Betadine)</td><td>MEDA Pharma</td><td>314997.8</td><td>Sterility actions</td></tr><tr><td>Ketamine 100 mg/ml (stock solution)</td><td>MERIAL</td><td></td><td>Anesthesic</td></tr><tr><td>Xylazine 100 mg/ml in PBS (stock solution)</td><td>Sigma</td><td>X1251-1G</td><td>Anesthesic and muscle relaxant</td></tr><tr><td>Buprenorphin 0.3 mg/ml stock solution</td><td>AXIENCE</td><td></td><td>Morphinic analgesic</td></tr><tr><td>Ophtalmic gel (0.2% cyclosporin)</td><td>Schering-Plough Animal Health</td><td></td><td>Eye protection</td></tr><tr><td>DPBS (+ CaCl&lt;sub&gt;2&lt;/sub&gt;, MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>GIBCO Life Technology</td><td>14040-174</td><td>to isolate embryos</td></tr><tr><td>HBSS Hanks&#039; Balanced Salt Solution (+ CaCl&lt;sub&gt;2&lt;/sub&gt;, MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>GIBCO Life Technology</td><td>24020-091</td><td>to wash out the blood and dissect the embryos</td></tr><tr><td>OPTI-MEM I GlutaMAX I</td><td>GIBCO Life Technology</td><td>51985-026</td><td>Medium for cultures</td></tr><tr><td>Fetal calf serum</td><td>EUROBIO</td><td>CVFSVF00-0U</td><td>additive for cultures</td></tr><tr><td>Penicilin and streptomycin</td><td>GIBCO Life Technology</td><td>15640-055</td><td>Antibiotics for cultures</td></tr><tr><td>2-Mercaptoethanol</td><td>GIBCO Life Technology</td><td>31350010</td><td>additive for cultures</td></tr><tr><td>LEICA MZ6 Dissection microscope</td><td>LEICA</td><td>MZ6 10445111</td><td>Occular W-Pl10x/23</td></tr><tr><td>Cold lamp source</td><td>SCHOTT VWR</td><td>KL1500 compact</td><td>Two goose neck fibers adapted</td></tr><tr><td>Silicone elastomer</td><td>World Precision Instruments</td><td>SYLG184</td><td>Dissecting Pad</td></tr><tr><td>Spoon, round and perforated</td><td>Fine Science Tools</td><td>10370-18</td><td>Dissection tools</td></tr><tr><td>Fine iris scissors</td><td>Fine Science Tools</td><td>14090-09</td><td>Dissection tools</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15018-10</td><td>Dissection tools</td></tr><tr><td>Forceps: Dumont #5/45 Inox 11 cm</td><td>F.S.T.</td><td>11253-25</td><td>Dissection tools</td></tr><tr><td>2 pairs of fine straight watchmakers&amp;rsquo; forceps Dumont #5 11 cm fine tips.</td><td>Fine Science Tools</td><td>11295-20</td><td>Dissection tools</td></tr><tr><td>Polyamid thread with needle 6-0 C-3 3/8c</td><td>ETHICON</td><td>F2403</td><td>for sutures</td></tr><tr><td>Needle-holder</td><td>MORIA/F.S.T.</td><td>12060-02</td><td>for sutures</td></tr><tr><td>Heating pad</td><td>VWR</td><td>100229-100</td><td>To maintain mouse temperature during anesthesy</td></tr><tr><td>Membrane Isopore RTTPO2500</td><td>DUTSCHER</td><td>44210</td><td>For FTOC</td></tr><tr><td>Terasaki 60 wells plates</td><td>FISHER</td><td>1x270 10318801</td><td>For hanging drop technique</td></tr><tr><td>Gauze swabs steriles 7.5 x 7.5 cm</td><td>Hydrex</td><td>11522</td><td>To apply disinfecting solution</td></tr><tr><td>Fluorescence or biotin labelled antibodies</td><td>BD Biosciences, Biolegend or e-Biocsiences&amp;nbsp;</td><td>Clone Number see table below</td><td>Staining cells</td></tr><tr><td>MACS Columns/Streptavidin Microbeads</td><td>Miltenyi Biotec</td><td>130-042-401/130-048-101</td><td>Cell depletion</td></tr><tr><td>Mice</td><td>Charles Rivers Laboratories (CD45.1) and Janvier Labs&amp;nbsp; (CD45.2)</td><td>C57BL/6 CD45.1 OR CD45.2</td><td>Source of cells and thymic lobes</td></tr><tr><td>Mice</td><td>CDTA Orl&amp;eacute;ans, France</td><td>CD 3 epsilon Ko CD45.2</td><td>Grafting experiment</td></tr></tbody></table>

characterization of thymic settling progenitors in the mouse embryo using in vivo and in vitro assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

In order to choose a method to deplete thymic lobes of endogenous thymocytes allowing the best development of colonizing progenitors, we compared the levels of T cell reconstitution in thymic lobes colonized after irradiation or a 5-day deoxy-guanosine (d-Gua) treatment. The results show that while there is no difference at day 9 of culture, irradiated lobes contained more T cells than those treated with d-Gua, at day 12. Thus, irradiation is more appropriate than d-Gua treatment to obtain T cell development after thymic...

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

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

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

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

characterization-thymic-settling-progenitors-mouse-embryo-using-vivo

Research

JoVE Journal

Developmental Biology

8.3K Views.  Institut Cochin. This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny. 

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

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

Biology

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Immunology and Infection

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Cancer Research

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Recreating the Thymic Microenvironment with iPSC-Derived Organoids

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Author Spotlight: Advancing Thymic Epithelial Cells and T-Cell Research with Human Thymic Organoids

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

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

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