Method Article

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

DOI:

10.3791/66093

⸱

November 10th, 2023

 ,  ,  ,  ,  ,  , 
1Wilmot Cancer Institute, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester, 3Department of Biomedical Genetics, University of Rochester Medical Center, 4Genomics Research Center, University of Rochester Medical Center, 5Division of Endocrinology and Metabolism, Department of Medicine, University of Rochester Medical Center

In This Article

Summary

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Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

Abstract

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The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Introduction

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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 hematopoi....

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Protocol

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All animal experiments were conducted in accordance with protocols approved by the University of Rochester University Committee on Animal Resources. Mice were bred and maintained in the animal care facilities at the University of Rochester. To model high-risk MDS, the commercially available NHD13 murine model19 is employed. In this model, bone marrow stromal cells are analyzed in female NHD13 mice at 8 weeks of age, before disease onset. De novo AML is generated as previously described6,11,20. The oncogenes used to induce AML, such as MLL-AF9 ....

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Results

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This article describes a flow cytometry-based method for analyzing bone marrow microenvironmental populations, such as the endothelial and mesenchymal stromal cells, from MDS and leukemia murine models (Figure 1). Figure 2 depicts the gating strategy for detection of populations of interest, beginning with the selection of cells (P1) in the digested and CD45/Ter119 depleted fraction through forward and side scatter profile. Example gating of cells in a leukemia .......

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Discussion

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Murine leukemia models have been extensively used to identify cell intrinsic and niche-driven signals that promote aggressive myeloid leukemia progression6,19,21. Here, a comprehensive flow cytometry-based protocol to define the cellular composition of the bone marrow microenvironment in murine models of MDS and AML is presented.

Prior to acquiring flow cytometric data from experimental samples, it is.......

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Disclosures

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No conflicts of interest declared.

Acknowledgements

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We would like to thank the URMC Flow Cytometry Core. This work was supported by American Society of Hematology Scholar Award, Leukemia Research Foundation award and NIH grants R01DK133131 and R01CA266617 awarded to J.B.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1 mL pipette Tips Genesee Scientific 24-165RL
1.7 mL Microcentrifuge TubesAVANTL211511-CS
10 µL pipette TipsGenesee Scientific 24-140RL
10 mL Individually Wrapped Sterile Serological PipettesGlobe scientific1760
1000 mL Vacuum Filtration FlaskNEST344021
15 mL Centrifuge TubeVWR10026-076
2 mL Aspirating PipetteNEST325011
200 µL pipette TipsGenesee Scientific 24-150-RL
25 mL Individually Wrapped Sterile Serological PipettesGlobe scientific1780
5 mL Individually Wrapped Sterile Serological PipettesGlobe scientific1740
5 mL Polystyrene Round-Bottom Tube  12 x 75 mm styleFalcon352054
5 mL Polystyrene Round-Bottom Tube with Cell Strainer Cap 12 x 75 mm styleFalcon352235
50 mL Centrifuge TubeNEST602052
6 Well, Flat Bottom with Low Evaporation LidFalcon353046
Absorbent Underpads with Waterproof Moisture BarrierVWR56616-031
APC MicroBeadsMiltenyi 130-090-855
autoMACS Pro SeparatorMiltenyi Biotec GmBH4425745
BD Pharmingen Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)BD Biosciences5531410.5 mg/mL 
Bovine Serum AlbuminSigma-AldrichA790666.000 g/mol
Brilliant Violet 421 anti-mouse Ly-6A/E (Sca-1) Antibody (D7)Invitrogen404-59810.2 mg/mL
C57BL/6J MiceJackson Laboratory 664
Carbon Dioxide Gas TankAirgasCD50
CD31 (PECAM-1) Monoclonal Antibody (390), PE-Cyanine7Invitrogen25-0311-820.2 mg/mL
CD45 Monoclonal Antibody (30-F11), APCInvitrogen17-0451-820.2 mg/mL
Cell Strainer 70 µm Nylon Falcon352350
Cole-Parmer Essentials Mortar and Pestle; Agate, 125 mLCole-ParmerEW-63100-62
Collagenase from Clostridium histolyticumSigma-AldrichC5138-500MG
Collagenase Type ISTEMCELL7415
Corning Mini CentrifugeCORNING6770
Corning Stripettor Ultra Pipet ControllerCorning4099
Deoxyribonuclease I from bovine pancreasSigma-AldrichD4513
Dispase II, powderGibco117105041
DPBS 10xgibco14200-075
eBioscience 1x RBC Lysis BufferInvitrogen00-4333-57
Ethanol absolute, KOPTEC, meets analytical specification of BP, Ph. Eur., USP (200 Proof)VWR89125-174
Fine scissors - sharpFine Science Tools14061-10
Foundation B Fetal Bovine SerumGeminiBio900-208
Gilson PIPETMAN L Pipette Starter KitsFisherScientific F167370G
Graefe ForcepsFine Science Tools11051-10
Hank's Balanced Salt Solution (HBSS) 10xgibco14185-052
HemocytometerFisher02-671-10
Incubator BINDERC150-UL
KimwipesKIMTECHK222101
LABGARD Class II, Type A2 Biological Safety CabinetNuaireNU-425-400
LD ColumnsMiltenyi Biotec GmBH130-042-901
LSE Vortex MixerCORNING6775
LSRII/Fortessa/Symphony A1Becton, Dickinson and Company647800L6
MACS MULTI STAND Miltenyi Biotec GmBH130-042-303
MACsmix Tube Rotator Miltenyi Biotec GmBH130-090-753
mIgGMillipore-Sigma18765-10mg2 mg/mL 
Nup98-HoxD13 (NHD13) MiceJackson Laboratory 010505
PE anti-mouse CD51 Antibody (RMV-7)Biolegend1041060.2 mg/mL
PE/Cyanine5 anti-mouse CD140a Antibody (RUO)Biolegend1359200.2 mg/mL
Penicillin-Streptomycin Gibco1514012210,000 U/mL
Plastipak 3 mL SyringeBecton, Dickinson and Company309657
Propidium Iodide - 1.0 mg/mL Solution in WaterThermoFisher ScientificP3566
QuadroMACS  Separator Miltenyi Biotec GmBH130-090-976
Sorvall X Pro / ST Plus Series CentrifugeThermo Scientific 75009521
TER-119 Monoclonal Antibody (TER-119), APCInvitrogen17-5921-820.2 mg/mL
Trypan Blue Solution 0.4%Gibco15-250-061
Ultrapure 0.5 M EDTA, pH 8.0 Invitrogen15575-038

References

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  1. Morrison, S. J., Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature. 505 (7483), 327-334 (2014).
  2. Boulais, P. E., Frenette, P. S. Making sense of hematopoietic stem cell niches. Blood. 125 (17), 2621-2629 (2015).
  3. Pinho, S., Frenette, P. S.

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Tags

Bone Marrow MicroenvironmentMyelodysplastic SyndromeAcute Myeloid LeukemiaMesenchymal Stromal CellsFlow CytometryMagnetic SeparationEndothelial CellsOsteolineage CellsHematopoietic Stem CellsCell Population Isolation

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