O.A.-W. is supported by an NIH K08 Clinical Investigator Award (1K08CA160647-01), a U.S. Department of Defense Postdoctoral Fellow Award in Bone Marrow Failure Research (W81XWH-12-1-0041), the Josie Roberston Investigator Program, and a Damon Runyon Clinical Investigator Award with Support from the Evan&#8217;s Foundation.

このプロトコルで説明されているすべての動物の手順は、実験動物の管理と使用に関する指針に従って実施された、メモリアルスローンケタリングがんセンターの施設内動物管理使用委員会（IACUCs）により承認された。 大腿骨から骨髄の1。吸引（BM）細胞<ol><li>精密気化器を用いて投与イソフルラン（1-4％）で大腿骨髄穿刺を受けるマウスを麻酔。麻酔の深さは、外科的処置および/または耳、足指、および尾部ピンチに関連付けられた呼吸数に変化がないことを観察することによって、プロシージャ中のあらゆる5〜10分を監視すべきである。麻酔はイソフルランで誘導し、また、イソフルランによる手順を通して維持されている。 0.05〜0.1ミリグラムの用量でブプレノルフィンの先制事前手続き線量は/ kgを皮下に、すべての6から12時間は、ADJのような手順に関連する痛みを防止するために使用することができますカルプロフェンにUNCT。 </li><li>角膜の乾燥を防ぐために、麻酔の導入後のマウスの眼に眼軟膏を適用します。 </li><li>毛皮を慎重に意図された吸引部位の面積よりも約150％大きく皮膚の領域から切り取られます。緩い毛皮は、湿ったガーゼパッドを用いて除去する。手順の間に低体温症を防ぐために、循環温水パッドまたは他の安全なサーモスタット制御された表面上でマウスを保管してください。 </li><li> （ポビドンヨード（ベタジン）またはクロルヘキシジン（Nolvasan）スクラブと70％イソプロピルアルコール、70％エタノールに浸したガーゼスポンジのいずれかと交互に）スクラブが交互に3組の吸引を受ける大腿骨を含む足全体を消毒する。 </li><li> BMを吸引する前に、滅菌リン酸緩衝生理食塩水（PBS）で洗浄：;：0.5ミリリットルツベルクリンシリンジ（27.5 Gゲージ0.5ccの体積）を濡らす。のPBS 200〜500μlの注射器を記入し、すぐに、PBSを追放。 2〜3トンにこの手順を繰り返しますのIME。 </li><li>薬指や小指のいずれかで脛骨を押すことによって大腿骨から脛骨を曲げておく。適切な麻酔面が達成されたことを確認してください。シリンジを親指と人差し指を使用して保持される。これは、顆を露出させることを可能にし、針の挿入を容易にする。 </li><li>針が大腿骨の2顆の間でセキュアに提出されるように、注射器を湿らせた後、膝蓋腱を通して針を挿入します。親指と人​​差し指で、大腿骨の骨端に近い骨幹を保持することによって、針は簡単に大腿骨のシャフトに挿入されている。 </li><li>それは大腿骨の軸に平行になるように外向き、上向き針を旋回さ。このアクションは、大腿骨シャフトからの骨髄の内容の検索を容易にします。 </li><li>大腿骨髄腔にゆっくりと押しながら、針を時計回りと反時計回りに回します。優しくSYを移動して、針の正確な位置決めを確認横方向にringe。 </li><li> BMキャビティ内に前後に針を移動させながら、優しく、負圧にする、後針プランジャーを引く。注：BM吸引量は、一般的に0.4〜0.8×10 6の単核細胞に相当し、約5μLになります。成功吸引は注射器の基部に針の上での血液の出現によって、視覚的に確認される。まだ血液がシリンジ内に見られない場合、それは小さな骨または組織片が針に貼り付けられている可能性がある。これは、（これはシリンジBM吸引する前にPBSで予め充填されるべきである1つの理由で）PBSで上下プランジャを移動させることにより、針から除去することができる。組織がシリンジから外れできない場合は、新しい針と注射器を（再びのPBS 200〜500μlの注射器を濡らし）を使用します。 </li><li> BMが正常大腿骨から吸引された後、大腿骨やマウスから針と注射器を取り外します。 </li><li>マイクロチューブPRに吸引BMを移動500μlのPBSでefilled。ほとんどのアプリケーションでは、BM細胞を、可能であればさらなる処理まで氷上で保存する。 </li><li>手続きの完了に続いて、5 mg / kgの皮下カルプロフェンと鎮痛剤を投与し。その後、麻酔からマウスを削除し、完全に回復するまで加熱パッドの上に置きます。注記：正しく行われれば吸引手順に従って経験しない合併症や苦痛があってはならない。 </li><li>住宅エリアにマウスを返す前に、彼らが歩き回ると食料や水に到達できることを確認してください。次の24時間での苦痛や感染後の手続きの兆候マウスを観察します。兆候としては、以下が挙げられる：定数出血、貧血、無気力を。これらの徴候のいずれかが後の手順を見ている場合は、動物（S）は、安楽死させなければならない。注：BM吸引/サンプリングを繰り返すことができるが、反復手順は同じ脚に繰り返される外傷を防ぐために反対側の大腿骨に対して行われるべきである。 FRに関する入手可能な情報はほとんどないBM吸引を行うことができるequency。大腿骨髄穿刺は、一般的にこれ以上の頻度で2週間ごとよりも繰り返される。 </li></ol>吸引しBM細胞における細胞含有量の2。評価<ol><li> 4 O Cまたは室温で5分間300×gで遠心分離することによりマイクロチューブ内のBM吸引や場所から検索された細胞をペレット化。 </li><li>上清を吸引してから、ACK赤血球細胞溶解バッファー（「塩化アンモニウムカリウム「溶解バッファー）500μl中にペレットを再懸濁します。 </li><li> 10分間、赤血球溶解緩衝液中で細胞をインキュベートし、次いで1mlのPBSを添加し、4°Cの 、またはRTで5分間300×gで再び混合物をスピンダウンする。注意：赤血球を溶解ペレットは現在のBM単核細胞で構成されています。これらは、（屠殺したマウスから採取BM細胞が使用されるのと同様に）FACS染色、細胞計数、移植、サイトスピン分析および/または任意の他の使用のために再懸濁させることができる。 </li></ol>

<ol><li>Sundberg, R., Hodgson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aspiration+of+bone+marrow+in+laboratory+animals%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Aspiration of bone marrow in laboratory animals.</a> Blood. 4, 557-561 (1949).</li>
<li>Schmitz, M., Bourquin, J. -P., Bornhauser, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+technique+for+intrafemoral+injection+and+bone+marrow+sampling+in+mouse+transplant+models%5BTitle%5D)+AND+%22Leukemia+%26+lymphoma%22%5BJournal%5D)">Alternative technique for intrafemoral injection and bone marrow sampling in mouse transplant models.</a> Leukemia & lymphoma. 52, 1806-1808 (2011).</li>
<li>Warner, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+evidence+for+cooperating+genetic+events+in+the+leukemic+transformation+of+normal+human+hematopoietic+cells%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells.</a> Leukemia. 19, 1794-1805 (2005).</li>
<li>Chiu, P. P., Jiang, H., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukemia-initiating+cells+in+human+T-lymphoblastic+leukemia+exhibit+glucocorticoid+resistance%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Leukemia-initiating cells in human T-lymphoblastic leukemia exhibit glucocorticoid resistance.</a> Blood. 116, 5268-5279 (2010).</li>
<li>Guan, Y., Gerhard, B., Hogge, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection%2C+isolation%2C+and+stimulation+of+quiescent+primitive+leukemic+progenitor+cells+from+patients+with+acute+myeloid+leukemia+%28AML%29%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML).</a> Blood. 101, 3142-3149 (2003).</li>
<li>Nolta, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+gold+standard+improves%3A+a+better+assay+for+HSCs%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">The gold standard improves: a better assay for HSCs.</a> Blood. 106, 1141-1142 (2005).</li>
<li>Kiel, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li>
<li>Purton, L., Scadden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limiting+factors+in+murine+hematopoietic+stem+cell+assays%5BTitle%5D)+AND+%22Cell+stem+cell%22%5BJournal%5D)">Limiting factors in murine hematopoietic stem cell assays.</a> Cell stem cell. 1, 263-270 (2007).</li>
<li>Dykstra, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+propagation+of+distinct+hematopoietic+differentiation+programs+in+vivo%5BTitle%5D)+AND+%22Cell+stem+cell%22%5BJournal%5D)">Long-term propagation of distinct hematopoietic differentiation programs in vivo.</a> Cell stem cell. 1, 218-229 (2007).</li>
<li>Jordan, C., Lemischka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+and+systemic+analysis+of+long-term+hematopoiesis+in+the+mouse%5BTitle%5D)+AND+%22Genes+%26+development%22%5BJournal%5D)">Clonal and systemic analysis of long-term hematopoiesis in the mouse.</a> Genes & development. 4, 220-232 (1990).</li>
<li>Challen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dnmt3a+is+essential+for+hematopoietic+stem+cell+differentiation%5BTitle%5D)+AND+%22Nature+genetics%22%5BJournal%5D)">Dnmt3a is essential for hematopoietic stem cell differentiation.</a> Nature genetics. 44, 23-31 (2012).</li>
<li>Hess, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selection+based+on+CD133+and+high+aldehyde+dehydrogenase+activity+isolates+long-term+reconstituting+human+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells.</a> Blood. 107, 2162-2169 (2006).</li>
<li>Mazurier, F., Doedens, M., Gan, O. I., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+myeloerythroid+repopulation+after+intrafemoral+transplantation+of+NOD-SCID+mice+reveals+a+new+class+of+human+stem+cells%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.</a> Nat Med. 9, 959-963 (2003).</li>
</ol>

著者らは、開示することは何もありません。

シリアルのBM吸引は、ヒトの血液疾患の臨床研究に不可欠な日常的な手順である。長時間の実験を通して、BMの細胞組成および成分の特徴付けマウスでは、BMの類似シリアルサンプリングを実行する能力は、同様に非常に貴重なものです。この手順では、マウスを犠牲にせずにも、末梢血の内容はBMに存在する細胞が反射性でなくてもよい場合においてBM中の追加の細胞型の存在を検出するため...

All blood cells are derived from hematopoietic stem cells (HSCs). Procedures which allow access to the bone marrow (BM), the site of the vast majority of HSC&#8217;s in mice and men, without requiring sacrifice of animals, provide a resource to monitor the source of hematopoiesis serially. The overall goal of the procedure described here is to provide a detailed protocol for the sampling of BM cells from the femur of live mice which provides material representative of the cells which would be retrieved by conventional harvest of BM cells through sacrifice of the mice. The advantage of this procedure over conventional harvest of BM cells is that this procedure does not require the sacrifice of mice and therefore allows for longitudinal study of the bone marrow compartment of mice over time. 
Although this murine bone marrow aspiration procedure has been used in a number of studies and described previously (see Sundberg et al. for a historical review1), formal step-by-step procedures illustrating this technique have not been previously published. This protocol enables routine serial sampling of the BM for purposes such as assessing engraftment in the BM of cells exogenously introduced into the mouse (as would be done in xenograft studies for instance2,3), analysis of chimerism in the BM for comparison with that of the peripheral blood (where results are not necessarily congruent), monitoring the abundance of specific cell types normally enriched in the marrow such as hematopoietic stem and progenitor cells, and retrieval of BM cells for ex vivo culture and/or transplantation. In addition, evaluation of marrow contents may be very useful in murine models of hematological disease as aspiration is helpful in assessing hematological disease burden at time points when disease may not be evident in the peripheral blood3-5. Moreover, this technique may be used to evaluate response of hematologic disease in the marrow to drugs administered in therapeutic studies. Thus, the procedure described here is ideal for investigators wishing to obtain access to bone marrow hematopoietic cells from mice for cytological analysis, flow cytometric analysis, in vitro culturing, and/or in vivo transplantation studies without requiring sacrifice or harm to the mice. 
The protocol for sampling of BM described here is quite similar to the technique used for intra-femoral injection of material directly into the marrow cavity3,6. The key difference being that this protocol details the procedure for removal of cells from the marrow as opposed to instilling material directly into the marrow cavity space as is done with intrafemoral injection.

<table><tbody><tr><td>PBS</td><td>PAA</td><td>H15-002</td><td></td></tr><tr><td>Bovine serum albumin</td><td>PAA</td><td>K41-001</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Homemade</td><td></td><td>in 1 L. Adjust pH 7.2 ~ 7.4 and filter sterile with 0.22 &amp;mu;m vacuum filter.</td></tr><tr><td>8.3 g Ammonium chloride</td><td>Fisher Scientific</td><td>A661-500</td><td></td></tr><tr><td>1 g Potassium bicarbonate</td><td>Fisher Scientific</td><td>P184-500</td><td></td></tr><tr><td>200 &amp;mu;l 0.5 M EDTA pH 8</td><td>Gibco</td><td>15575-038</td><td></td></tr><tr><td>RPMI 1640</td><td>PAA</td><td>E15-842</td><td></td></tr><tr><td>0.5 ml Tuberculin syringe 27.5 G</td><td>Becton Dickinson</td><td>305620</td><td></td></tr><tr><td>Sterile cell strainer 70 &amp;mu;m</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Attane</td><td>NDC:66794-014-25</td><td></td></tr><tr><td>Blunt-end needle</td><td>Stemcell Technologies</td><td>28110</td><td></td></tr><tr><td>PrecisionGlide needle 23 G</td><td>Becton Dickinson</td><td>305193</td><td></td></tr><tr><td>3 ml Syringe Luer-Lok tip</td><td>Becton Dickinson</td><td>309657</td><td></td></tr><tr><td>Non-tissue culture treated plate, 6 Well</td><td>Becton Dickinson</td><td>351146</td><td></td></tr><tr><td>12 x 75 mm 5 ml tubes&amp;nbsp;</td><td>Becton Dickinson</td><td>352054</td><td>FACS staining</td></tr><tr><td>12 x 75 mm 5 ml tubes with cell-strainer cap</td><td>Becton Dickinson</td><td>352235</td><td>FACS staining</td></tr><tr><td>NK1.1 APC-Cy7</td><td>Biolegend</td><td>108723</td><td></td></tr><tr><td>CD11b APC-Cy7</td><td>Biolegend</td><td>101225</td><td></td></tr><tr><td>CD45R (B220) APC-Cy7</td><td>Biolegend</td><td>103223</td><td></td></tr><tr><td>CD3 APC-Cy7</td><td>Biolegend</td><td>100222</td><td></td></tr><tr><td>Ly-6G and Ly-6C (Gr-1) APC-Cy7</td><td>Biolegend</td><td>108423</td><td></td></tr><tr><td>Ter119 APC-Cy7</td><td>Biolegend</td><td>116223</td><td></td></tr><tr><td>CD19 APC-Cy7</td><td>Biolegend</td><td>302217</td><td></td></tr><tr><td>CD4 APC-Cy7</td><td>BioLegend</td><td>317417</td><td></td></tr><tr><td>CD117 (c-KIT) PE</td><td>BioLegend</td><td>105808</td><td></td></tr><tr><td>Ly-6A/E (Sca-1) PE-Cy7</td><td>Biolegend</td><td>122513</td><td></td></tr><tr><td>CD34 APC</td><td>Biolegend</td><td>128612</td><td></td></tr><tr><td>CD16/32 e450</td><td>eBioscience</td><td>48-0161-82</td><td></td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma-Aldrich</td><td>32670</td><td></td></tr><tr><td>MethoCult GF M3434</td><td>STEMCELLTECHNOLOGIES</td><td>3434</td><td>For methocellulose culture</td></tr><tr><td>Carprofen</td><td>Crescent Chemical Company</td><td>C11045850</td><td>1 dose (5mg/kg)&amp;nbsp;</td></tr><tr><td>Flow cytometer, LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Puralube vet ointment (sterile petrolatum ophthalmic ointment)</td><td>Dechra-US</td><td>17033-211-38</td><td></td></tr></tbody></table>

femoral bone marrow aspiration in live mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

ライブC57/B6マウスの大腿BM吸引犠牲後の同じマウスのBM従来の収穫に続くBM単核細胞を得るために利用された。 2つの方法によって得られたBM単核細胞を、次いで、BM細胞（1）細胞学的分析によって分析し、（2）造血幹/前駆細胞の相対頻度（HSPCs）を決定し、ソートHSPCs（3）ex vivoで培養した。後者の実験では、系統陰性のSca1 + C-KIT +（LSK）細胞は、骨髄吸引によってだけでなく、従来のBM...

Watch this Scientific Journal Video about Femoral Bone Marrow Aspiration in Live Mice at JoVE.com

Femoral Bone Marrow Aspiration in Live Mice

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

生きたマウスにおける大腿骨髄吸引

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

femoral-bone-marrow-aspiration-in-live-mice

Research

JoVE Journal

Medicine

37.9K ビュー.  Memorial Sloan-Kettering Cancer Center. This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

ビデオ: Femoral Bone Marrow Aspiration in Live Mice

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

発生生物学

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

骨髄ニッチに造血細胞の生着のダイナミクスを研究する遺伝的モデルと生体内蛍光顕微鏡 (IVFM) を組み合わせること

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨転移モデル

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

マウス骨髄単球の追跡<em&gt;インビボ</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

免疫・感染学

骨髄性腫瘍における骨髄微小環境の単離と特性評価

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

著者スポットライト:マウス血液悪性腫瘍における骨髄微小環境の分析 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

骨髄異形成症候群および急性骨髄性白血病における骨髄微小環境集団の同定

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

がん研究

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

以下のための大腿骨のウィンドウチャンバーモデル<em&gt;インビボ</emマウス骨髄内&gt;細胞トラッキング

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

好中球系統細胞の質量細胞法解析に対する全骨髄の調製

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

クローン造造を研究するマウスにおける骨髄移植手順

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

生きたマウスにおける大腿骨髄吸引

要約

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造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

骨髄ニッチに造血細胞の生着のダイナミクスを研究する遺伝的モデルと生体内蛍光顕微鏡 (IVFM) を組み合わせること

骨転移モデル

マウス骨髄単球の追跡

骨髄異形成症候群および急性骨髄性白血病における骨髄微小環境集団の同定

骨髄異形成症候群および急性骨髄性白血病における骨髄微小環境集団の同定

骨髄異形成症候群および急性骨髄性白血病における骨髄微小環境集団の同定

以下のための大腿骨のウィンドウチャンバーモデル

好中球系統細胞の質量細胞法解析に対する全骨髄の調製

クローン造造を研究するマウスにおける骨髄移植手順