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 

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

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.

저자 스포트라이트: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies(쥐 혈액 악성 종양의 골수 미세환경 분석) 

골수이형성 증후군 및 급성 골수성 백혈병의 골수 미세환경 집단 식별

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   

골수성 백혈병 미세환경의 특성화를 위한 쥐 골수 채취 및 분리 

시작하려면 안락사된 해부된 쥐의 뼈에서 조직을 제거합니다. 세척된 뼈를 얼음 위에 놓인 FACS 완충액이 들어 있는 새로운 6웰 플레이트에 넣습니다. 뼈를 2-5 밀리리터의 FACS 완충액이 있는 모르타르에 옮깁니다.골수가 방출될 때까지 원을 그리며 뼈를 갈아줍니다. 3밀리리터 주사기로 골수를 위로 당겨 내려 균질화합니다. 균질액을 다른 주사기로 당긴 다음 70마이크로미터 셀 스트레이너를 통해 50밀리리터 튜브로 여과합니다.두 번째로 균질화하기 전에 FACS 완충액으로 필터의 조직 덩어리를 모르타르로 다시 헹굽니다. 이전과 같이 헹군 티슈를 걸러냅니다. 그런 다음 남은 뼈 스피큘을 FACS 완충액으로 모르타르에 다시 헹굽니다.그런 다음 최대 수율을 보장하기 위해 조각을 15밀리리터 튜브로 씻어냅니다. 골수를 소화하려면 먼저 300G에서 섭씨 4도에서 5분 동안 원심분리합니다. 골수 펠릿을 2 밀리리터의 소화 혼합물에 재현탁시킵니다.혼합물을 15밀리리터 튜브로 옮깁니다. 그런 다음 섭씨 37도에서 회전근에서 45분 동안 배양합니다. 이제 효소 분해를 중지하기 위해 10mL의 FACS 완충액을 추가합니다.혼합물을 70마이크로미터 셀 스트레이너를 통해 50밀리리터 튜브로 여과합니다. 서스펜션을 섭씨 4도에서 7분 동안 400G로 원심분리합니다. 펠릿을 1mL의 RBC 용해 완충액에 재현탁시킵니다.그런 다음 10mL의 FACS 버퍼를 추가하여 용해를 중단합니다. 그런 다음 이전과 같이 혼합물을 50밀리리터 튜브로 여과합니다. 마지막으로 혼합물을 섭씨 4도에서 5분 동안 300G로 원심분리합니다.상층액을 버린 후 펠릿을 100마이크로리터의 FACS 완충액에 재현탁시킵니다. 뼈 스피큘의 소화를 위해서는 먼저 뼈 스피큘을 소용돌이치게 합니다. 상층액을 디캔팅하고 뼈를 바닥에 유지합니다.다음으로, 뼈 미세 소화 혼합물 1m리터에 스피큘을 재현탁시킵니다. 섭씨 37도에서 60분 동안 회전근에서 튜브를 배양합니다. 마지막으로 10mL의 FACS 완충액으로 분해를 중지합니다.혼합물을 적혈구 용해 및 소화된 골수가 들어 있는 50밀리리터 튜브에 여과합니다.

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 ...