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 

Isolement et caractérisation du microenvironnement de la moelle osseuse dans les néoplasmes myéloïdes

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.

Pleins feux sur l’auteur : Analyse du microenvironnement de la moelle osseuse dans les hémopathies malignes murines 

Identification des populations microenvironnementales de moelle osseuse dans le syndrome myélodysplasique et la leucémie myéloïde aiguë

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   

Prélèvement et isolement de moelle osseuse murine pour la caractérisation du microenvironnement de la leucémie myéloïde 

Pour commencer, retirez le tissu des os de la souris euthanasiée disséquée. Placez les os nettoyés dans une nouvelle plaque à six puits contenant un tampon FACS placé sur de la glace. Transférez les os dans un mortier avec deux à cinq millilitres de tampon FACS.Broyez les os dans un mouvement circulaire jusqu’à ce que la moelle osseuse soit libérée. À l’aide d’une seringue de trois millilitres, tirez vers le haut et rincez la moelle osseuse pour l’homogénéiser. Tirez l’homogénat dans une autre seringue, puis filtrez-le à travers une crépine de 70 micromètres dans un tube de 50 millilitres.Rincez les morceaux de tissu de leur filtre dans le mortier avec le tampon FACS avant d’homogénéiser une deuxième fois. Filtrez le tissu rincé comme précédemment. Ensuite, rincez les spicules osseux restants dans le mortier avec un tampon FACS.Rincez ensuite les morceaux dans un tube de 15 millilitres pour assurer un rendement maximal. Pour digérer la moelle osseuse, centrifugez-la d’abord à 300 g pendant cinq minutes à quatre degrés Celsius. Remettre en suspension la pastille de moelle osseuse dans deux millilitres de mélange de digestion.Transférez le mélange dans un tube de 15 millilitres. Ensuite, incubez-le à 37 degrés Celsius pendant 45 minutes sur un rotateur. Maintenant, ajoutez 10 millilitres de tampon FACS pour arrêter la digestion enzymatique.Filtrez le mélange à travers une crépine cellulaire de 70 micromètres dans un tube de 50 millilitres. Centrifuger la suspension à 400 G pendant sept minutes à quatre degrés Celsius. Remettre la pastille en suspension dans un millilitre de tampon de lyse des globules rouges.Ensuite, ajoutez 10 millilitres de tampon FACS pour arrêter la lyse. Filtrez ensuite le mélange comme précédemment dans un tube de 50 millilitres. Enfin, centrifugez le mélange à 300 G pendant cinq minutes à quatre degrés Celsius.Après avoir éliminé le surnageant, remettre la pastille en suspension dans 100 microlitres de tampon FACS. Pour la digestion des spicules osseux, il faut d’abord vortex les spicules osseux. Décanter le surnageant et conserver les os au fond.Ensuite, remettez les spicules en suspension dans un millilitre du mélange de digestion des spicules osseux. Incuber le tube sur un rotateur pendant 60 minutes à 37 degrés Celsius. Enfin, arrêtez la digestion avec 10 millilitres de tampon FACS.Filtrer le mélange dans le tube de 50 millilitres contenant de la moelle osseuse lysée et digérée par les globules rouges.

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 ...