Name: Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment
Uploaded: 2023-11-10T13:00:00
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murine bone marrow harvest and isolation for the characterization of myeloid leukemia microenvironment 

Isolamento e caracterização do microambiente da medula óssea em neoplasias mieloides

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.

Autor em destaque: Analisando o microambiente da medula óssea em malignidades hematológicas murinas 

Identificação de Populações Microambientais de Medula Óssea na Síndrome Mielodisplásica e Leucemia Mielóide Aguda

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   

Coleta e isolamento de medula óssea murina para caracterização do microambiente da leucemia mielóide 

Para começar, remova o tecido dos ossos do camundongo dissecado eutanasiado. Coloque os ossos limpos em uma nova placa de seis poços contendo tampão FACS colocado no gelo. Transfira os ossos para uma argamassa com dois a cinco mililitros de tampão FACS.Triture os ossos em um movimento circular até que a medula óssea seja liberada. Com uma seringa de três mililitros, puxe para cima e lave a medula óssea para homogeneizá-la. Puxe o homogeneizado para outra seringa e, em seguida, filtre-o através de um filtro de células de 70 micrômetros em um tubo de 50 mililitros.Enxaguar os pedaços de tecido de seu filtro de volta para a argamassa com tampão FACS antes de homogeneizar uma segunda vez. Filtre o tecido enxaguado como antes. Em seguida, enxágue as espículas ósseas restantes de volta na argamassa com tampão FACS.Em seguida, lave as peças em um tubo de 15 mililitros para garantir o máximo rendimento. Para digerir a medula óssea, primeiro centrifuga-a a 300 G por cinco minutos a quatro graus Celsius. Ressuspenda a pelota de medula óssea em dois mililitros de mistura de digestão.Transfira a mistura para um tubo de 15 mililitros. Em seguida, incube-o a 37 graus Celsius por 45 minutos em um rotador. Agora, adicione 10 mililitros de tampão FACS para interromper a digestão enzimática.Filtre a mistura através de um filtro de células de 70 micrômetros em um tubo de 50 mililitros. Centrifugar a suspensão a 400 G durante sete minutos a quatro graus Celsius. Ressuspenda o pellet em um mililitro de tampão de lise eritrocitária.Em seguida, adicione 10 mililitros de tampão FACS para interromper a lise. Em seguida, filtre a mistura como antes em um tubo de 50 mililitros. Por fim, centrifugar a mistura a 300 G por cinco minutos a quatro graus Celsius.Após o descarte do sobrenadante, ressuspenda o pellet em 100 microlitros de tampão FACS. Para a digestão das espículas ósseas, primeiro, vórtice as espículas ósseas. Decantar o sobrenadante e reter os ossos na parte inferior.Em seguida, ressuspenda as espículas em um mililitro da mistura de digestão de espículas ósseas. Incubar o tubo em um rotador por 60 minutos a 37 graus Celsius. Finalmente, pare a digestão com 10 mililitros de tampão FACS.Filtrar a mistura para o tubo de 50 mililitros contendo a medula óssea lisada e digerida por hemácias.

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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 ...