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 

Aislamiento y caracterización del microambiente de la médula ósea en 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 destacado: Análisis del microambiente de la médula ósea en neoplasias hematológicas malignas murinas 

Identificación de poblaciones microambientales de médula ósea en el síndrome mielodisplásico y la leucemia mieloide 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   

Recolección y aislamiento de médula ósea murina para la caracterización del microambiente de la leucemia mieloide 

Para empezar, retira el tejido de los huesos del ratón diseccionado con eutanasia. Coloque los huesos limpios en una nueva placa de seis pocillos que contenga tampón FACS colocado en hielo. Transfiera los huesos a un mortero con dos a cinco mililitros de tampón FACS.Muele los huesos con movimientos circulares hasta que se libere la médula ósea. Con una jeringa de tres mililitros, tire hacia arriba y hacia abajo de la médula ósea para homogeneizarla. Coloque el homogeneizado en otra jeringa, luego fíltrelo a través de un colador celular de 70 micrómetros en un tubo de 50 mililitros.Enjuague los trozos de tejido de su filtro nuevamente en el mortero con tampón FACS antes de homogeneizar por segunda vez. Filtre el pañuelo enjuagado como antes. A continuación, enjuague las espículas óseas restantes nuevamente en el mortero con tampón FACS.A continuación, enjuague las piezas en un tubo de 15 mililitros para garantizar el máximo rendimiento. Para digerir la médula ósea, primero centrifugarla a 300 G durante cinco minutos a cuatro grados centígrados. Vuelva a suspender el gránulo de médula ósea en dos mililitros de mezcla de digestión.Transfiere la mezcla a un tubo de 15 mililitros. Luego incubarlo a 37 grados centígrados durante 45 minutos en un rotador. Ahora, agregue 10 mililitros de tampón FACS para detener la digestión enzimática.Filtre la mezcla a través de un colador de celdas de 70 micrómetros en un tubo de 50 mililitros. Centrifugar la suspensión a 400 G durante siete minutos a cuatro grados centígrados. Vuelva a suspender el gránulo en un mililitro de tampón de lisis de glóbulos rojos.A continuación, agregue 10 mililitros de tampón FACS para detener la lisis. Luego filtre la mezcla como antes en un tubo de 50 mililitros. Por último, centrifugar la mezcla a 300 G durante cinco minutos a cuatro grados centígrados.Después de desechar el sobrenadante, vuelva a suspender el gránulo en 100 microlitros de tampón FACS. Para la digestión de las espículas óseas, primero, vórtice las espículas óseas. Decantar el sobrenadante y conservar los huesos en la parte inferior.A continuación, vuelva a suspender las espículas en un mililitro de la mezcla de digestión de la espícula ósea. Incubar el tubo en un rotador durante 60 minutos a 37 grados centígrados. Finalmente, detenga la digestión con 10 mililitros de tampón FACS.Filtre la mezcla en el tubo de 50 mililitros que contiene la médula ósea lisada y digerida por glóbulos rojos.

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 ...