Name: generation of lymph node-fat pad chimeras for the study of lymph node stromal cell origin
Uploaded: 2013-12-16T13:45:00
Description: Watch this Scientific Journal Video about Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin at JoVE.com

We are grateful to the personnel of the Biomedical Services Unit of the University of Birmingham for taking care of our animal colonies. This work was supported by the EU FP7 integrated project INFLACARE to JC.

Mice were bred and maintained under SPF conditions in the Biomedical Service Unit at the University of Birmingham according to UK Home Office and local ethics committee regulations. All procedures described in this protocol are covered under a Project License approved by both local ethics committee and the Home Office.
1. Isolation of Newborn LNs
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	<li>Sacrifice the newborn mice by cervical dislocation.</li>
	<li>Section the head, and open the body with scissors from the top of the thora...

<ol>
	<li>Mueller, S. N., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+contributions+to+the+homeostasis+and+functionality+of+the+immune+system.">Stromal cell contributions to the homeostasis and functionality of the immune system.</a> Nat. Rev. Immunol. 9, 618-629 (2009).</li><li>Roozendaal, R., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell-immune+cell+interactions.">Stromal cell-immune cell interactions.</a> Annu. Rev. Immunol. 29, 23-43 (2011).</li><li>Turley, S. J., Fletcher, A. L., Elpek, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stromal+and+haematopoietic+antigen-presenting+cells+that+reside+in+secondary+lymphoid+organs.">The stromal and haematopoietic antigen-presenting cells that reside in secondary lymphoid organs.</a> Nat. Rev. Immunol. 10, 813-825 (2010).</li><li>Katakai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizer-like+reticular+stromal+cell+layer+common+to+adult+secondary+lymphoid+organs.">Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs.</a> J. Immunol. 181, 6189-6200 (2008).</li><li>Link, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblastic+reticular+cells+in+lymph+nodes+regulate+the+homeostasis+of+naive+T+cells.">Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.</a> Nat. Immunol. 8, 1255-1265 (2007).</li><li>Benezech, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+stromal+organizer+cells+during+lymph+node+development.">Ontogeny of stromal organizer cells during lymph node development.</a> J. Immunol. 184, 4521-4530 (2010).</li><li>Cupedo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presumptive+lymph+node+organizers+are+differentially+represented+in+developing+mesenteric+and+peripheral+nodes.">Presumptive lymph node organizers are differentially represented in developing mesenteric and peripheral nodes.</a> J. Immunol. 173, 2968-2975 (2004).</li><li>Avan de Pavert, S., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+development+of+lymphoid+tissues.">New insights into the development of lymphoid tissues.</a> Nat. Rev. Immunol. 10, 664-674 (2010).</li><li>Vondenhoff, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTbetaR+signaling+induces+cytokine+expression+and+up-regulates+lymphangiogenic+factors+in+lymph+node+anlagen.">LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen.</a> J. Immunol. 182, 5439-5445 (2009).</li><li>Dougall, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANK+is+essential+for+osteoclast+and+lymph+node+development.">RANK is essential for osteoclast and lymph node development.</a> Genes Dev. 13, 2412-2424 (1999).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+peripheral+lymph+node+genesis+by+the+tumor+necrosis+factor+family+member+TRANCE.">Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE.</a> J. Exp. Med. 192, 1467-1478 (2000).</li><li>Carragher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stroma-derived+defect+in+NF-kappaB2-/-+mice+causes+impaired+lymph+node+development+and+lymphocyte+recruitment.">A stroma-derived defect in NF-kappaB2-/- mice causes impaired lymph node development and lymphocyte recruitment.</a> J. Immunol. 173, 2271-2279 (2004).</li><li>White, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin+a-dependent+and+-independent+signals+regulate+stromal+organizer+cell+homeostasis+during+lymph+node+organogenesis.">Lymphotoxin a-dependent and -independent signals regulate stromal organizer cell homeostasis during lymph node organogenesis.</a> Blood. 110, 1950-1959 (2007).</li><li>Benezech, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin-beta+receptor+signaling+through+NF-kappaB2-RelB+pathway+reprograms+adipocyte+precursors+as+lymph+node+stromal+cells.">Lymphotoxin-beta receptor signaling through NF-kappaB2-RelB pathway reprograms adipocyte precursors as lymph node stromal cells.</a> Immunity. 37, 721-734 (2012).</li><li>Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-positive+epithelium+and+mesenchyme+cells+are+both+required+for+T-cell+development+in+the+thymus.">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>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J. Vis. Exp. (9), e404 (2007).</li><li>Krautler, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+dendritic+cells+emerge+from+ubiquitous+perivascular+precursors.">Follicular dendritic cells emerge from ubiquitous perivascular precursors.</a> Cell. 150, 194-206 (2012).</li></ol>

In this article we presented a method to assay and quantify the contribution of adipose tissue progenitor cells to the developing LNs and two techniques that allow the analysis of their progeny. Dissection of embryonic fat pads and newborns LNs are delicate and require manual skills gained by a lot of practice prior to the generation of the actual LN-fat pad chimera. To control the quality of the dissections, flow-cytometric analysis can be performed on the fat pads and LNs. Embryonic fat pad preparation should be...

Lymph nodes (LNs) are key organs of the immune system situated at strategic sites in the body, along the lymphatic vasculature network. They enable filtration of antigens and pathogens and provide a site for antigen presentation to lymphocytes and induction of adaptive immune responses. The stroma, which forms the basic structure of the LN and orchestrates the movement of the different hematopoietic participants of the adaptive immune response, is central to the function of these organs. Different populations of stromal cells supply essential and specific cues for the movements, localization, survival, proliferation and maturation of the hematopoietic component of the immune system1-3. Adult LN stromal cells fall in three categories: the blood endothelial cells, the lymphatic endothelial cells and fibroblasts. These three categories encompass heterogeneous populations. The fibroblastic populations contain fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), marginal reticular cells (MRC), while the fibroblasts forming the capsule, the medulla and other cells which are not yet identified1-5. The origins and the mechanisms governing the maturation of the different LN stromal cell populations are unclear and the absence of specific markers allowing the fate mapping of specific LN stromal cell populations rends their study particularly difficult. However, a full understanding of the ontogeny of LN stromal cells is necessary to the comprehension of adaptive immune responses, the mechanisms contributing to tolerance and is at the basis of the development of artificial LNs.
So far, the study of LN stromal cell origin and development has been mostly limited to the direct assessment of LN development in embryos and newborns in wild type mice and different knockout mouse strains6-9. These approaches are limited by the embryonic and perinatal lethality of some of the mouse strains carrying deletions in genes important for LN development. Moreover, some of the genes essential for lymphoid tissue development are also involved in a wide range of biological process as it is the case for RANK10-11 or NF-&#954;B212. To address these issues, isolation and transplantation of embryonic LNs under the kidney capsule of a host mouse have been performed12-13. This technique allows, for example, the transfer of genetically modified embryonic LNs in a wild type environment to assess organ development and the recruitment and organization of host cells. However, the growth of embryonic LN grafted under the kidney capsule of an adult host is impaired, thus limiting the use of this technique.
LNs and fat deposits are anatomically closely associated and they develop simultaneously during embryogenesis. We recently demonstrated that the association LN/adipose tissue plays a crucial role in the provision of stromal cell progenitors for the LNs. In particular, signaling through the LT&#946;R controls the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting maturation towards a LN stromal cell phenotype14. Here we describe a method based on generation of LN-fat pad chimeras allowing the tracing of adipose tissue derived cells in the developing LN. This method will be useful to determine the contribution of adipose tissues to different LN stromal cell populations and combined with the use of tissues from genetically modified mouse strains, will allow a better understanding of the mechanisms controlling the differentiation of the different LN stromal cell subsets.

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	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mm Sterilin Petri Dish</td>
			<td>Appleton Woods</td>
			<td>SC265</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Sterilin Petri Dish</td>
			<td>Appleton Woods</td>
			<td>SC260</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive slides</td>
			<td>Surgipath</td>
			<td>00202</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD31 eFluor 450</td>
			<td>eBioscience</td>
			<td>48-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD4 Alexa 647</td>
			<td>eBioscience</td>
			<td>51-0041</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-0451</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgM Rhodamine Red</td>
			<td>Stratech</td>
			<td>715-296-020-JIR</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Podoplanin PE/Cy7</td>
			<td>Biolegend</td>
			<td>127411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Podoplanin purified</td>
			<td>eBioscience</td>
			<td>14-5381</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps Inox Biologie</td>
			<td>FST</td>
			<td>11252-20</td>
			<td>Extra fine forceps for embryonic and newborn dissections</td>
		</tr>
		<tr>
			<td>EDTA solution 0.5 M</td>
			<td>Sigma</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>ERTR-7</td>
			<td>Biogenesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened fine iris scissors straight 11 cm</td>
			<td>FST</td>
			<td>14090-11</td>
			<td>Small scissors for dissection</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopore Membrane Filters 0.8 &#956;m ATTP</td>
			<td>Millipore</td>
			<td>ATTPO 1300</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential Amino Acid Solution (100x)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic box with lid</td>
			<td>Watkins and Doncaster</td>
			<td>E6052</td>
			<td>Sandwich box for in vitro organ culture. Drill two holes in the lid to allow gas exchange in the CO2 incubator.</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Sigma</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Sponges Vulkan Underwrap</td>
			<td>Patterson Medical</td>
			<td>004383</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>LEICA MZ95</td>
			<td>Dissecting microscope with zoom</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5436</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of lymph node-fat pad chimeras for the study of lymph node stromal cell origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Three weeks after transplantation, the LN/fat pad chimera is recovered from the kidney. The chimera is now very similar to a normal LN in its own fat pad, and the LN is visible in the center of the adipose tissue (Figure 1). If the LN can&#39;t be identified, it is possible that it was lost during the transplantation on the kidney. When assessing the role of genes potentially important in LN development, the LNs recovered may remain very small and more difficult to find. This is the case when adipose tis...

Watch this Scientific Journal Video about Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin at JoVE.com

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

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