Name: Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment
Uploaded: 2023-11-10T13:00:00
Description: Watch this Scientific Journal Video about Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment at JoVE.com

murine bone marrow harvest and isolation for the characterization of myeloid leukemia microenvironment 

骨髄性腫瘍における骨髄微小環境の単離と特性評価

The bone marrow microenvironment consists of hematopoietic cells, non-hematopoietic stromal cells, and the extracellular matrix1,2. This microenvironment can promote hematopoietic stem cell self-renewal, regulate lineage differentiation, and provide structural and mechanical support to the bone tissue1,2,3,4,5. The stromal niche includes osteolineage cells, fibroblasts, nerve cells, and endothelial cells6, while the hematopoietic niche consists of the lymphoid and myeloid populations1,2,3. In addition to supporting normal HSCs, the bone marrow microenvironment can also play a role in the development of hematopoietic stem cell disorders such as MDS and AML7,8,9,10,11. Mutations in osteolineage cells have been shown to promote the development of MDS, AML, and other myeloproliferative neoplasms10,12,13,14.
Myelodysplastic syndromes are a group of pre-leukemic disorders that arise from mutations in hematopoietic stem cells. MDS is frequently associated with a block in HSC differentiation and the production of dysplastic cells, which can often lead to bone marrow failure. MDS is the most commonly diagnosed myeloid neoplasm in the United States and is associated with a 3-year survival rate of 35%-45%15. MDS is often associated with a high risk of transformation to acute myeloid leukemia. This can be a fatal complication, as MDS-derived AML is resistant to most therapies and likely to relapse. AML that arises de novo due to translocations or mutations in hematopoietic stem and progenitors is also often resistant to standard chemotherapy16,17. Since MDS and AML are primarily diseases of the elderly, with the majority diagnosed over the age of 60 years, most patients are ineligible for curative bone marrow transplants. There is, thus, a significant need to identify novel regulators of disease progression. Since the bone marrow microenvironment can provide support for malignant cells14, defining changes in the bone marrow niche with disease progression may lead to the identification of novel therapeutics aimed at inhibiting tumor niche remodeling. There is, therefore, a significant need to identify novel regulators of disease progression. To this end, it is critical to identify and characterize changes in the bone marrow stromal cell populations that may provide support for the malignant cells.
Several murine models of AML and MDS have been generated and can be used to study changes in the bone marrow microenvironment during disease initiation and progression6,1,19,20,21,22. Here, protocols to identify changes in the bone marrow stromal cell populations using murine models of retrovirally induced AML6,20, as well as the commercially available Nup98-HoxD13 (NHD13) model of high-risk MDS to AML transformation19, are described. Mice transplanted with de novo AML cells succumb to the disease in 20-30 days6. The NHD13 mice develop cytopenias and bone marrow dysplasia around 15-20 weeks, which eventually transforms into AML, and nearly 75% of the mice succumb to the disease around 32 weeks. To analyze the murine model bone marrow microenvironment populations, bones are harvested, bone marrow and bone spicules are digested using enzymatic digestion, and the cells are then enriched for CD45-/Ter119- non-hematopoietic populations by magnetic sorting. While similar analyses have been previously described11,13,22,23,24,25, they often focus on either the bone marrow or the bone and do not incorporate cells from both sources in their analyses. The combined characterization of these populations, in conjunction with gene expression analyses, can provide a comprehensive understanding of how the cellular hematopoietic microenvironment provides support for disease initiation and progression (Figure 1). While the protocol described below focuses on retrovirally induced AML model and a genetic MDS model, these strategies can be easily adapted to study changes in the bone marrow niche of any murine model of interest.

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 

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

Our research aims to explore how the bone marrow's changing environment influences tumor growth. We developed a flow cytometry protocol to analyze the non-immune cells in bone marrow and bone tissues, focusing on murine hematological malignancies. This method is adaptable for studying bone marrow in various murine models.Our study focuses on isolating and identifying alterations in bone marrow stromal populations during cancer progression. Understanding their impact on disease onset and advancement could pave the way for new treatments that target these cancer-facilitating changes in the bone marrow stromal cells. Our protocol uniquely combines mechanical and enzymatic digestions, magnetic depletions, and flow cytometry to delineate the bone marrow microenvironment cellular makeup.It stands out by employing magnetic separation and incorporating cells from both bone and marrow, offering a more comprehensive representation of this microenvironment.

Watch this Scientific Journal Video about Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment at JoVE.com

Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment   

骨髄性白血病微小環境の特性評価のためのマウス骨髄採取と単離 

まず、安楽死させた解剖マウスの骨から組織を取り除きます。洗浄した骨を、氷上に置かれたFACS緩衝液を含む新しい6ウェルプレートに入れます。骨を2〜5ミリリットルのFACS緩衝液を入れた乳鉢に移します。骨髄が放出されるまで、円を描くように骨をすりつぶします。3ミリリットルのシリンジで、骨髄を引き上げて洗い流し、均質化します。ホモジネートを別のシリンジに引き込み、70マイクロメートルのセルストレーナーでろ過して50ミリリットルのチューブに入れます。2回目の均質化を行う前に、フィルターからFACSバッファーで組織塊をモルタルに戻します。前と同じようにすすぎ組織をろ過します。次に、残りの骨棘をFACS緩衝液で乳鉢に洗い流します。次に、ピースを15ミリリットルのチューブに洗い流して、最大の収量を確保します。骨髄を消化するには、まず300 Gで摂氏4度で5分間遠心分離します。骨髄ペレットを2ミリリットルの消化混合物に再懸濁します。混合物を15ミリリットルのチューブに移します。その後、ローテーター上で摂氏37度で45分間インキュベートします。次に、10ミリリットルのFACSバッファーを添加して、酵素消化を停止します。混合物を70マイクロメートルのセルストレーナーで50ミリリットルのチューブにろ過します。懸濁液を400Gで摂氏4度で7分間遠心分離します。ペレットを1ミリリットルの赤血球溶解緩衝液に再懸濁します。次に、10ミリリットルのFACS緩衝液を添加して溶解を停止します。次に、前と同じように混合物を50ミリリットルのチューブにろ過します。最後に、混合物を300gで摂氏4度で5分間遠心分離します。上清を廃棄した後、ペレットを100マイクロリットルのFACS緩衝液に再懸濁します。骨棘の消化のために、まず、骨棘をボルテックスします。上澄みをデカントし、底に骨を残します。次に、棘を1ミリリットルの骨棘消化混合物に再懸濁します。チューブをローテーター上で摂氏37度で60分間インキュベートします。最後に、10ミリリットルのFACS緩衝液で消化を停止します。混合物を濾過し、溶解および消化した骨髄を含む50ミリリットルのチューブに入れます。

Research

JoVE Journal

Cancer Research

Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment 

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 ...