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 

髓系肿瘤中骨髓微环境的分离和表征

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.

作者聚焦：分析小鼠血液系统恶性肿瘤的骨髓微环境 

识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体

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.

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

鼠骨髓采集和分离用于骨髓性白血病微环境表征 

首先，从安乐死解剖小鼠的骨头中取出组织。将清洁后的骨头放入新的六孔板中，该板含有放置在冰上的FACS缓冲液。将骨头转移到装有 2 至 5 毫升 FACS 缓冲液的研钵中。以圆周运动研磨骨头，直到骨髓释放。用三毫升注射器，向上拉并冲洗骨髓以使其均质化。将匀浆拉入另一个注射器中，然后通过70微米的细胞过滤器将其过滤到50毫升的管中。在第二次均质化之前，用FACS缓冲液将组织块从过滤器冲洗回研钵中。像以前一样过滤冲洗过的组织。接下来，用FACS缓冲液将剩余的骨针冲洗回研钵中。然后将碎片冲洗到 15 毫升管中，以确保最大产量。为了消化骨髓，首先在4摄氏度下以300G离心五分钟。将骨髓沉淀重悬于两毫升消化混合物中。将混合物转移到 15 毫升管中。然后在旋转器上在 37 摄氏度下孵育 45 分钟。现在，加入 10 毫升 FACS 缓冲液以停止酶消化。通过70微米的细胞过滤器将混合物过滤到50毫升的管中。在4摄氏度下以400G离心悬浮液7分钟。将沉淀重悬于一毫升红细胞裂解缓冲液中。接下来，加入 10 毫升 FACS 缓冲液以停止裂解。然后像以前一样将混合物过滤到 50 毫升管中。最后，在4摄氏度下以300G离心混合物五分钟。弃去上清液后，将沉淀重悬于100μL FACS缓冲液中。为了消化骨针，首先，涡旋骨针。倒出上清液并保留底部的骨头。接下来，将针状物重悬于一毫升骨针状体消化混合物中。将试管在旋转器上在 37 摄氏度下孵育 60 分钟。最后，用10毫升FACS缓冲液停止消化。将混合物过滤到含有红细胞裂解和消化的骨髓的 50 毫升管中。

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 ...