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.

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 and NRas, are tagged with GFP or YFP, allowing for the analysis of the non-leukemic GFP- bone marrow populations using flow cytometry. In brief, 10-week-old female C57BL/6J mice are transplanted with murine GFP/YFP+ AML cells, and the bone marrow is harvested 2 weeks post-transplant. While female mice are used in this study for demonstration purposes, this protocol can be conducted with either male or female mice. It can also be carried out using either one femur or all long bones.
1. Bone marrow harvesting
NOTE: For details on the animal dissection protocol, please refer to Amend et al.26.
<ol>
	<li>Clean the mortar and pestle with 70% ethanol, rinse them with chilled FACS buffer (Table 1), and place them on ice to cool before starting the harvest. Also, place MACs buffer (Table 1) on the bench to allow it to reach room temperature.</li>
	<li>Euthanize the animal following the institutional animal care and use guidelines and protocols.</li>
	<li>On the benchtop, thoroughly spray the mouse with 70% ethanol until its fur is wet. Using forceps and curved scissors, lift the skin on the abdomen and make two incisions approximately 0.5 mm in length on both sides of the mouse, lateral from the abdomen. Next, make a 0.5 mm incision distal from the abdomen. Pull down to remove the skin and fur from the mouse&#39;s legs.</li>
	<li>Place scissors perpendicular to the pelvis, press down while pulling up on the femur with forceps. The femoral head should detach from the pelvis. Separate the femur and tibia at the patellofemoral joint. Place the bones in FACS buffer in a 6-well plate on ice.</li>
	<li>Remove tissue from the bones using laboratory-grade tissue and place the cleaned bones into a new 6-well plate with fresh FACS buffer on ice.</li>
	<li>Place all bones into the mortar with 2-5 mL of FACS buffer so that all bones are immersed in the buffer (adjust the volume of buffer based on how many bones you are processing). Crush and grind the bones using the pestle in a circular motion until the bone marrow tissue is released.</li>
	<li>Using a 3 mL syringe, homogenize the bone marrow by pulling up and flushing down the liquid from the mortar.</li>
	<li>Using a 3 mL syringe, pull up the liquid from the mortar and filter it through a 70 &#181;m cell strainer into a 50 mL tube on ice. Rinse the bone/tissue chunks from the filter back into the mortar with FACS buffer and return to step 1.7 to homogenize and strain a second time. This constitutes the bone marrow fraction.</li>
	<li>Rinse the remaining bone pieces (spicules) back into the mortar with FACS buffer and flush them into a 15 mL tube using FACS buffer to ensure maximum cell yield. This is the bone spicules fraction.</li>
</ol>
2. Digestion of bone marrow
<ol>
	<li>Centrifuge the bone marrow at 300 x g for 5 min at 4 &#176;C. Decant and discard the supernatant.</li>
	<li>Resuspend the bone marrow in 2 mL of the bone marrow digestion mixture (Table 1) and transfer it to a 15 mL tube. Incubate at 37 &#176;C for 45 min on a rotator.</li>
	<li>Add 10 mL of FACS buffer to stop the enzymatic digestion. Filter the mixture through a 70 &#181;m cell strainer into a new 50 mL tube.</li>
	<li>Pellet the mixture at 400 x g for 7 min at 4 &#176;C.</li>
	<li>Resuspend the bone marrow pellet in 1 mL of RBC lysis buffer (see Table of Materials). Incubate for 4 min on ice.</li>
	<li>Add 10 mL of FACS buffer to stop the lysis. Filter the mixture through a 70 &#181;m cell strainer into a new 50 mL tube.</li>
	<li>Pellet the mixture at 300 x g for 5 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 100 &#181;L of FACS buffer.</li>
</ol>
3. Digestion of bone spicules
<ol>
	<li>Vortex the bone spicules from step 1.9 and allow them to settle. Decant the supernatant and retain the bone at the bottom.</li>
	<li>Resuspend the bone spicules in 1 mL of the bone spicule digestion mixture (Table 1).</li>
	<li>Place the tubes on a tube rotator for 60 min at 37 &#176;C.</li>
	<li>Add 10 mL of FACS buffer to stop the enzymatic digestion. Filter the mixture through a 70 &#181;m cell strainer into the 50 mL tube containing RBC-lysed and digested bone marrow.</li>
</ol>
4. Staining
<ol>
	<li>Gently mix the bone spicules and bone marrow cell suspension.</li>
	<li>Use 10 &#181;L of the cell suspension to count the live cell number on a hemocytometer using 0.4% Trypan blue-based staining, as described in published protocols27. Collect 50,000 cells for an unstained control from the cell suspension.</li>
	<li>Centrifuge the remaining cells at 300 x g for 5 min at 4 &#176;C. Remove the supernatant and resuspend them in 100 &#181;L of FACS buffer. 
	NOTE: Antibodies can be titrated to determine the ideal dilution. Antibody selection (epitopes and fluorochromes) can be customized.</li>
	<li>For staining with antibodies for magnetic depletion, add FC Block (1 &#181;L per 25 x 106 cells), CD45-APC (10 &#181;L per 25 x 106 cells), and Ter119-APC (4 &#181;L per 25 x 106 cells) (see Table of Materials).</li>
	<li>Incubate on ice for 20 min. Wash with FACS buffer, remove 50,000 cells (~50 &#181;L) for the APC-stained control, centrifuge at 300 x g for 5 min at 4 &#176;C, and resuspend in 100 &#181;L of FACS buffer.</li>
	<li>For staining of the cell suspension with microbeads for magnetic depletion, add mIgG (8 &#181;L per 25 x 106 cells) and Anti-APC microbeads (20 &#181;L per 25 x 106 cells) (see Table of Materials).</li>
	<li>Incubate on ice for 20 min. Wash with 10 mL FACS buffer, centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
</ol>
5. Depletion of sample by magnetic sorting
NOTE: This step is carried out using a commercially available manual magnetic separator according to the manufacturer&#39;s instructions. This step can also be performed with an automated separator (see Table of Materials).
<ol>
	<li>Prepare the LD column by washing it with 2 mL of MACs buffer (Table 1). Discard the effluent and change the collection tube.</li>
	<li>Resuspend up to 1 x 108 cells in MACs buffer and filter them through a 5 mL test tube with a 35 &#181;m cell strainer cap.</li>
	<li>Place the LD column on the magnetic separator stand. Position a 5 mL test tube below the column to collect the eluate.</li>
	<li>Add the cell suspension to the prepared LD column. Allow the negative fraction to flow through into the collection tube. Wash the column twice with 1 mL of MACs buffer, collecting the eluate in the same tube. This is the negative fraction used in step 5.6 below.</li>
	<li>Remove the LD column from the magnetic separator stand and place it on a new 5 mL test tube. Use a pipette to dispense 3 mL of buffer into the column to flush out cells that have been positively labeled, using the column plunger.</li>
	<li>Centrifuge the negative and positive fractions at 300 x g for 5 min at 4 &#176;C. Resuspend them in 100 &#181;L of FACS buffer.</li>
	<li>Count 10 &#181;L of the negative and positive fractions with 0.4% Trypan blue. The volumes for the osteo-analysis/endothelial panel staining below will be based on this cell count.</li>
	<li>Use 50,000 live cells from the positive fraction for flow cytometry gating controls.</li>
</ol>
6. Osteo-analysis/endothelial panel stain
NOTE: Compensation should be performed following standard flow cytometry protocols, including all appropriate staining and gating controls.
<ol>
	<li>For staining of the CD45/Ter119 negative fraction (1 &#181;L of each antibody per 1 x 106 cells), add CD31-PE-Cy7, Sca-1-BV421, CD51-PE, and CD140a-PE-Cy5 (see Table of Materials).</li>
	<li>Incubate on ice for 20 min. Wash with 2 mL of FACS buffer, then centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Add 1 mL of FACS buffer and a 1:1000 dilution of PI for live/dead staining, then filter the sample through a 5 mL test tube with a 35 &#181;m cell strainer cap.</li>
	<li>Analyze the cells on a multi-color flow cytometer.</li>
</ol>

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

<ol>
	<li>Morrison, S. J., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+marrow+niche+for+haematopoietic+stem+cells.">The bone marrow niche for haematopoietic stem cells.</a> Nature. 505 (7483), 327-334 (2014).</li><li>Boulais, P. E., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+sense+of+hematopoietic+stem+cell+niches.">Making sense of hematopoietic stem cell niches.</a> Blood. 125 (17), 2621-2629 (2015).</li><li>Pinho, S., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cell+activity+and+interactions+with+the+niche.">Haematopoietic stem cell activity and interactions with the niche.</a> Nat Rev Mol Cell Biol. 20 (5), 303-320 (2019).</li><li>Kfoury, Y., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cell+contributions+to+the+stem+cell+niche.">Mesenchymal cell contributions to the stem cell niche.</a> Cell Stem Cell. 16 (3), 239-253 (2015).</li><li>Itkin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+bone+marrow+blood+vessels+differentially+regulate+haematopoiesis.">Distinct bone marrow blood vessels differentially regulate haematopoiesis.</a> Nature. 532 (7599), 323-328 (2016).</li><li>Bajaj, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cd98-mediated+adhesive+signaling+enables+the+establishment+and+propagation+of+acute+myelogenous+leukemia.">Cd98-mediated adhesive signaling enables the establishment and propagation of acute myelogenous leukemia.</a> Cancer Cell. 30 (5), 792-805 (2016).</li><li>Konopleva, M. Y., Jordan, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia+stem+cells+and+microenvironment:+Biology+and+therapeutic+targeting.">Leukemia stem cells and microenvironment: Biology and therapeutic targeting.</a> J Clin Oncol. 29 (5), 591-599 (2011).</li><li>Kim, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+notch+activation+in+microenvironment+leads+to+myeloproliferative+disease.">Defective notch activation in microenvironment leads to myeloproliferative disease.</a> Blood. 112 (12), 4628-4638 (2008).</li><li>Walkley, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microenvironment-induced+myeloproliferative+syndrome+caused+by+retinoic+acid+receptor+gamma+deficiency.">A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency.</a> Cell. 129 (6), 1097-1110 (2007).</li><li>Kode, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukaemogenesis+induced+by+an+activating+&#946;-catenin+mutation+in+osteoblasts.">Leukaemogenesis induced by an activating &#946;-catenin mutation in osteoblasts.</a> Nature. 506 (7487), 240-244 (2014).</li><li>Hanoun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myelogenous+leukemia-induced+sympathetic+neuropathy+promotes+malignancy+in+an+altered+hematopoietic+stem+cell+niche.">Acute myelogenous leukemia-induced sympathetic neuropathy promotes malignancy in an altered hematopoietic stem cell niche.</a> Cell Stem Cell. 15 (3), 365-375 (2014).</li><li>Raaijmakers, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+progenitor+dysfunction+induces+myelodysplasia+and+secondary+leukaemia.">Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia.</a> Nature. 464 (7290), 852-857 (2010).</li><li>Frisch, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+inhibition+of+osteoblastic+cells+in+an+in+vivo+mouse+model+of+myeloid+leukemia.">Functional inhibition of osteoblastic cells in an in vivo mouse model of myeloid leukemia.</a> Blood. 119 (2), 540-550 (2012).</li><li>Bajaj, J., Diaz, E., Reya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+cancer+initiation+and+progression.">Stem cells in cancer initiation and progression.</a> J Cell Biol. 219 (1), e201911053 (2020).</li><li>Sekeres, M. A., Taylor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+myelodysplastic+syndromes:+A+review.">Diagnosis and treatment of myelodysplastic syndromes: A review.</a> Jama. 328 (9), 872-880 (2022).</li><li>Zeisig, B. B., Kulasekararaj, A. G., Mufti, G. J., So, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snapshot:+Acute+myeloid+leukemia.">Snapshot: Acute myeloid leukemia.</a> Cancer Cell. 22 (5), 698-698.e1 (2012).</li><li>Krivtsov, A. V., Armstrong, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mll+translocations,+histone+modifications+and+leukaemia+stem-cell+development.">Mll translocations, histone modifications and leukaemia stem-cell development.</a> Nat Rev Cancer. 7 (11), 823-833 (2007).</li><li>Yoshimi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+alterations+in+rna+splicing+and+epigenetic+regulation+drive+leukaemogenesis.">Coordinated alterations in rna splicing and epigenetic regulation drive leukaemogenesis.</a> Nature. 574 (7777), 273-277 (2019).</li><li>Lin, Y. W., Slape, C., Zhang, Z., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nup98-hoxd13+transgenic+mice+develop+a+highly+penetrant,+severe+myelodysplastic+syndrome+that+progresses+to+acute+leukemia.">Nup98-hoxd13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia.</a> Blood. 106 (1), 287-295 (2005).</li><li>Kwon, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraspanin+3+is+required+for+the+development+and+propagation+of+acute+myelogenous+leukemia.">Tetraspanin 3 is required for the development and propagation of acute myelogenous leukemia.</a> Cell Stem Cell. 17 (2), 152-164 (2015).</li><li>Bajaj, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+genome-wide+crispr+screen+identifies+the+rna-binding+protein+staufen2+as+a+key+regulator+of+myeloid+leukemia.">An in vivo genome-wide crispr screen identifies the rna-binding protein staufen2 as a key regulator of myeloid leukemia.</a> Nat Cancer. 1 (4), 410-422 (2020).</li><li>Krivtsov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+from+committed+progenitor+to+leukaemia+stem+cell+initiated+by+mll-af9.">Transformation from committed progenitor to leukaemia stem cell initiated by mll-af9.</a> Nature. 442 (7104), 818-822 (2006).</li><li>Baryawno, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cellular+taxonomy+of+the+bone+marrow+stroma+in+homeostasis+and+leukemia.">A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia.</a> Cell. 177 (7), 1915-1932.e16 (2019).</li><li>Tikhonova, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+marrow+microenvironment+at+single-cell+resolution.">The bone marrow microenvironment at single-cell resolution.</a> Nature. 569 (7755), 222-228 (2019).</li><li>Balderman, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+bone+marrow+microenvironment+improves+outcome+in+a+murine+model+of+myelodysplastic+syndrome.">Targeting of the bone marrow microenvironment improves outcome in a murine model of myelodysplastic syndrome.</a> Blood. 127 (5), 616-625 (2016).</li><li>Amend, S. R., Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hind+limb+long+bone+dissection+and+bone+marrow+isolation.">Murine hind limb long bone dissection and bone marrow isolation.</a> JoVE. 110, e53936 (2016).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+Using+a+Hemacytometer+to+Count+Cells.">Science Education Database. Basic Methods in Cellular and Molecular Biology. Using a Hemacytometer to Count Cells.</a> , (2023).</li><li>Passaro, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascular+permeability+in+the+bone+marrow+microenvironment+contributes+to+acute+myeloid+leukemia+progression+and+drug+response.">Increased vascular permeability in the bone marrow microenvironment contributes to acute myeloid leukemia progression and drug response.</a> Blood. 128 (22), 2662 (2016).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+factor+is+selectively+secreted+by+arterial+endothelial+cells+in+bone+marrow.">Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow.</a> Nat Commun. 9 (1), 2449 (2018).</li><li>Baccin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+single-cell+and+spatial+transcriptomics+reveal+the+molecular,+cellular+and+spatial+bone+marrow+niche+organization.">Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization.</a> Nat Cell Biol. 22 (1), 38-48 (2020).</li><li>Ebrahimi Dastgurdi, M., Ejeian, F., Nematollahi, M., Motaghi, A., Nasr-Esfahani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+digestion+strategies+on+characteristics+and+differentiation+potential+of+human+dental+pulp+stem+cells.">Comparison of two digestion strategies on characteristics and differentiation potential of human dental pulp stem cells.</a> Arch Oral Biol. 93, 74-79 (2018).</li><li>Abreu-Velez, A. M., Howard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+IV+in+normal+skin+and+in+pathological+processes.">Collagen IV in normal skin and in pathological processes.</a> N Am J Med Sci. 4 (1), 1-8 (2012).</li></ol>

No conflicts of interest declared.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL pipette Tips&#160;</td>
			<td>Genesee Scientific&#160;</td>
			<td>24-165RL</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL Microcentrifuge Tubes</td>
			<td>AVANT</td>
			<td>L211511-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L pipette Tips</td>
			<td>Genesee Scientific&#160;</td>
			<td>24-140RL</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Individually Wrapped Sterile Serological Pipettes</td>
			<td>Globe scientific</td>
			<td>1760</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Vacuum Filtration Flask</td>
			<td>NEST</td>
			<td>344021</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>VWR</td>
			<td>10026-076</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Aspirating Pipette</td>
			<td>NEST</td>
			<td>325011</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette Tips</td>
			<td>Genesee Scientific&#160;</td>
			<td>24-150-RL</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Individually Wrapped Sterile Serological Pipettes</td>
			<td>Globe scientific</td>
			<td>1780</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Individually Wrapped Sterile Serological Pipettes</td>
			<td>Globe scientific</td>
			<td>1740</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube&#160; 12 x 75 mm style</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube with Cell Strainer Cap 12 x 75 mm style</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well, Flat Bottom with Low Evaporation Lid</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent Underpads with Waterproof Moisture Barrier</td>
			<td>VWR</td>
			<td>56616-031</td>
			<td></td>
		</tr>
		<tr>
			<td>APC MicroBeads</td>
			<td>Miltenyi&#160;</td>
			<td>130-090-855</td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Pro Separator</td>
			<td>Miltenyi Biotec GmBH</td>
			<td>4425745</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>0.5 mg/mL&#160;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>66.000 g/mol</td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-mouse Ly-6A/E (Sca-1) Antibody (D7)</td>
			<td>Invitrogen</td>
			<td>404-5981</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jackson Laboratory&#160;</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide Gas Tank</td>
			<td>Airgas</td>
			<td>CD50</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 (PECAM-1) Monoclonal Antibody (390), PE-Cyanine7</td>
			<td>Invitrogen</td>
			<td>25-0311-82</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (30-F11), APC</td>
			<td>Invitrogen</td>
			<td>17-0451-82</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#181;m Nylon&#160;</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cole-Parmer Essentials Mortar and Pestle; Agate, 125 mL</td>
			<td>Cole-Parmer</td>
			<td>EW-63100-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C5138-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>STEMCELL</td>
			<td>7415</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Mini Centrifuge</td>
			<td>CORNING</td>
			<td>6770</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Stripettor Ultra Pipet Controller</td>
			<td>Corning</td>
			<td>4099</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>D4513</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II, powder</td>
			<td>Gibco</td>
			<td>117105041</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 10x</td>
			<td>gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience 1x RBC Lysis Buffer</td>
			<td>Invitrogen</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute, KOPTEC, meets analytical specification of BP, Ph. Eur., USP (200 Proof)</td>
			<td>VWR</td>
			<td>89125-174</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors - sharp</td>
			<td>Fine Science Tools</td>
			<td>14061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Foundation B Fetal Bovine Serum</td>
			<td>GeminiBio</td>
			<td>900-208</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson&#160;PIPETMAN L Pipette Starter Kits</td>
			<td>FisherScientific&#160;</td>
			<td>F167370G</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS) 10x</td>
			<td>gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>BINDER</td>
			<td>C150-UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>KIMTECH</td>
			<td>K222101</td>
			<td></td>
		</tr>
		<tr>
			<td>LABGARD Class II, Type A2 Biological Safety Cabinet</td>
			<td>Nuaire</td>
			<td>NU-425-400</td>
			<td></td>
		</tr>
		<tr>
			<td>LD Columns</td>
			<td>Miltenyi Biotec GmBH</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>LSE Vortex Mixer</td>
			<td>CORNING</td>
			<td>6775</td>
			<td></td>
		</tr>
		<tr>
			<td>LSRII/Fortessa/Symphony A1</td>
			<td>Becton, Dickinson and Company</td>
			<td>647800L6</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MULTI STAND&#160;</td>
			<td>Miltenyi Biotec GmBH</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MACsmix Tube Rotator&#160;</td>
			<td>Miltenyi Biotec GmBH</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>mIgG</td>
			<td>Millipore-Sigma</td>
			<td>18765-10mg</td>
			<td>2 mg/mL&#160;</td>
		</tr>
		<tr>
			<td>Nup98-HoxD13 (NHD13) Mice</td>
			<td>Jackson Laboratory&#160;</td>
			<td>010505</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD51 Antibody (RMV-7)</td>
			<td>Biolegend</td>
			<td>104106</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>PE/Cyanine5 anti-mouse CD140a Antibody (RUO)</td>
			<td>Biolegend</td>
			<td>135920</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>10,000 U/mL</td>
		</tr>
		<tr>
			<td>Plastipak 3 mL Syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide - 1.0 mg/mL Solution in Water</td>
			<td>ThermoFisher Scientific</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS&#160; Separator&#160;</td>
			<td>Miltenyi Biotec GmBH</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall X Pro / ST Plus Series Centrifuge</td>
			<td>Thermo Scientific&#160;</td>
			<td>75009521</td>
			<td></td>
		</tr>
		<tr>
			<td>TER-119 Monoclonal Antibody (TER-119), APC</td>
			<td>Invitrogen</td>
			<td>17-5921-82</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 0.5 M EDTA, pH 8.0&#160;</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 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.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

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

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.

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 ...

Watch this Scientific Journal Video about Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies at JoVE.com

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.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

identifying-bone-marrow-microenvironmental-populations

Research

JoVE Journal

Cancer Research

812 Views.  University of Rochester Medical Center. 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 an...

Video: Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment 

To begin, remove the tissue from the bones of the euthanized dissected mouse. Place the cleaned bones into a new six-well plate containing FACS buffer placed on ice. Transfer the bones into a mortar with two to five milliliters of FACS buffer.Grind the bones in a circular motion until the bone marrow is released. With a three milliliter syringe, pull up and flush down the bone marrow to homogenize it. Pull the homogenate into another syringe, then filter it through a 70 micrometer cell strainer into a 50 milliliter tube.Rinse the tissue chunks from their filter back into the mortar with FACS buffer before homogenizing a second time. Filter the rinsed tissue as before. Next, rinse the remaining bone spicules back into the mortar with FACS buffer.Then flush the pieces into a 15 milliliter tube to ensure maximum yield. To digest the bone marrow, first centrifuge it at 300 G for five minutes at four degrees Celsius. Resuspend the bone marrow pellet in two milliliters of digestion mixture.Transfer the mixture to a 15 milliliter tube. Then incubate it at 37 degrees Celsius for 45 minutes on a rotator. Now, add 10 milliliters of FACS buffer to stop the enzymatic digestion.Filter the mixture through a 70 micrometer cell strainer into a 50 milliliter tube. Centrifuge the suspension at 400 G for seven minutes at four degrees Celsius. Resuspend the pellet in one milliliter of RBC lysis buffer.Next, add 10 milliliters of FACS buffer to halt the lysis. Then filter the mixture as before into a 50 milliliter tube. Lastly, centrifuge the mixture at 300 G for five minutes at four degrees celsius.After discarding the supernatant, resuspend the pellet in 100 microliters of FACS buffer. For the digestion of bone spicules, first, vortex the bone spicules. Decant the supernatant and retain the bones at the bottom.Next, resuspend the spicules in one milliliter of the bone spicule digestion mixture. Incubate the tube on a rotator for 60 minutes at 37 degrees Celsius. Finally, stop the digestion with 10 milliliters of FACS buffer.Filter the mixture into the 50 milliliter tube containing RBC lysed and digested bone marrow.

Murine Bone Marrow Staining for the Characterization of Myeloid Leukemia Microenvironment

To begin, mix the isolated murine bone marrow suspension with the bone spicule suspension. Pipette 10 microliters of the cell suspension onto a hemocytometer for counting. Centrifuge the cell suspension at 300 G for five minutes at four degrees Celsius.Then resuspend the pellet in 100 microliters of FACS buffer. To stain the cells with antibodies for magnetic depletion, add FC block, CD45 APC, and TER-119 APC. Then wash the cell suspension with FACS buffer.After centrifuging the remaining mixture, resuspend the pellet in 100 microliters of FACS buffer. To stain the cell suspension with microbeads for magnetic depletion, add mouse IgG and anti-APC microbeads. Incubate the mixture on ice for 20 minutes.Wash the LD column with two milliliters of MACS buffer. Resuspend the cells in the buffer, then filter it through a five milliliter test tube with a 35 micrometer cell strainer cap. Next, set the LD column on the magnetic separator stand and place a five milliliter test tube under the column to collect the eluted material.Pipette the cell suspension into the LD column and collect the negative fraction flow through in the test tube. Wash the column two times with one milliliter of MACS buffer and collect the eluent material in the same tube. Place the LD column over a new five milliliter test tube.With a pipette, dispense three milliliters of buffer into the column, then flush out the positively labeled cells into a five milliliter test tube. Centrifuge both the positive and negative fractions, then resuspend the pellet in 100 microliters of FACS buffer. To stain the CD45 TER-119 negative fraction add one microliter of each antibody for every 10 to the sixth cells.Place the suspension on ice for 20 minutes. Then wash the suspension with two milliliters of FACS buffer before centrifuging. Add one milliliter of the buffer to the pellet and pipette propidium iodide for live cell staining.Finally, filter the sample through a 35 micrometer cell strainer into a five milliliter test tube before analyzing the cells on a multicolor flow cytometer. Arteriolar endothelial cells significantly expanded in the acute myeloid leukemia microenvironment with a concomitant loss in the sinusoidal endothelial populations. A small expansion in the mesenchymal stromal cells were seen at eight weeks of age.