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

Summary

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

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

The bone marrow microenvironment consists of hematopoietic cells, non-hematopoietic stromal cells, and the extracellular matrix^1,2. This microenvironment can promote hematopoietic stem cell self-renewal, regulate lineage differentiation, and provide structural and mechanical support to the bone tissue^1,2,3,4,5. The stromal niche includes osteolineage cells, fibroblasts, nerve cells, and endothelial cells⁶, while the hematopoietic niche consists of the lymphoid and myeloid populations^1,2,3. 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 AML^7,8,9,10,11. Mutations in osteolineage cells have been shown to promote the development of MDS, AML, and other myeloproliferative neoplasms^10,12,13,14.

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 chemotherapy^16,17. 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 progression^{6,1,19,20,21,22}. Here, protocols to identify changes in the bone marrow stromal cell populations using murine models of retrovirally induced AML^6,20, 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 described^{11,13,22,23,24,25}, 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.

Protocol

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 described^6,11,20. 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.

1. 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.
2. Euthanize the animal following the institutional animal care and use guidelines and protocols.
3. 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's legs.
4. 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.
5. 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.
6. 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.
7. Using a 3 mL syringe, homogenize the bone marrow by pulling up and flushing down the liquid from the mortar.
8. Using a 3 mL syringe, pull up the liquid from the mortar and filter it through a 70 µm 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.
9. 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.

2. Digestion of bone marrow

1. Centrifuge the bone marrow at 300 x g for 5 min at 4 °C. Decant and discard the supernatant.
2. 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 °C for 45 min on a rotator.
3. Add 10 mL of FACS buffer to stop the enzymatic digestion. Filter the mixture through a 70 µm cell strainer into a new 50 mL tube.
4. Pellet the mixture at 400 x g for 7 min at 4 °C.
5. Resuspend the bone marrow pellet in 1 mL of RBC lysis buffer (see Table of Materials). Incubate for 4 min on ice.
6. Add 10 mL of FACS buffer to stop the lysis. Filter the mixture through a 70 µm cell strainer into a new 50 mL tube.
7. Pellet the mixture at 300 x g for 5 min at 4 °C. Remove the supernatant and resuspend the pellet in 100 µL of FACS buffer.

3. Digestion of bone spicules

1. Vortex the bone spicules from step 1.9 and allow them to settle. Decant the supernatant and retain the bone at the bottom.
2. Resuspend the bone spicules in 1 mL of the bone spicule digestion mixture (Table 1).
3. Place the tubes on a tube rotator for 60 min at 37 °C.
4. Add 10 mL of FACS buffer to stop the enzymatic digestion. Filter the mixture through a 70 µm cell strainer into the 50 mL tube containing RBC-lysed and digested bone marrow.

4. Staining

1. Gently mix the bone spicules and bone marrow cell suspension.
2. Use 10 µL 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.
3. Centrifuge the remaining cells at 300 x g for 5 min at 4 °C. Remove the supernatant and resuspend them in 100 µL of FACS buffer.
   NOTE: Antibodies can be titrated to determine the ideal dilution. Antibody selection (epitopes and fluorochromes) can be customized.
4. For staining with antibodies for magnetic depletion, add FC Block (1 µL per 25 x 10⁶ cells), CD45-APC (10 µL per 25 x 10⁶ cells), and Ter119-APC (4 µL per 25 x 10⁶ cells) (see Table of Materials).
5. Incubate on ice for 20 min. Wash with FACS buffer, remove 50,000 cells (~50 µL) for the APC-stained control, centrifuge at 300 x g for 5 min at 4 °C, and resuspend in 100 µL of FACS buffer.
6. For staining of the cell suspension with microbeads for magnetic depletion, add mIgG (8 µL per 25 x 10⁶ cells) and Anti-APC microbeads (20 µL per 25 x 10⁶ cells) (see Table of Materials).
7. Incubate on ice for 20 min. Wash with 10 mL FACS buffer, centrifuge at 300 x g for 5 min at 4 °C.

5. Depletion of sample by magnetic sorting

NOTE: This step is carried out using a commercially available manual magnetic separator according to the manufacturer's instructions. This step can also be performed with an automated separator (see Table of Materials).

1. Prepare the LD column by washing it with 2 mL of MACs buffer (Table 1). Discard the effluent and change the collection tube.
2. Resuspend up to 1 x 10⁸ cells in MACs buffer and filter them through a 5 mL test tube with a 35 µm cell strainer cap.
3. Place the LD column on the magnetic separator stand. Position a 5 mL test tube below the column to collect the eluate.
4. 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.
5. 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.
6. Centrifuge the negative and positive fractions at 300 x g for 5 min at 4 °C. Resuspend them in 100 µL of FACS buffer.
7. Count 10 µL 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.
8. Use 50,000 live cells from the positive fraction for flow cytometry gating controls.

6. Osteo-analysis/endothelial panel stain

NOTE: Compensation should be performed following standard flow cytometry protocols, including all appropriate staining and gating controls.

1. For staining of the CD45/Ter119 negative fraction (1 µL 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).
2. Incubate on ice for 20 min. Wash with 2 mL of FACS buffer, then centrifuge at 300 x g for 5 min at 4 °C.
3. 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 µm cell strainer cap.
4. Analyze the cells on a multi-color flow cytometer.

Results

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 ...

Discussion

Murine leukemia models have been extensively used to identify cell intrinsic and niche-driven signals that promote aggressive myeloid leukemia progression^6,19,21. 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...

Materials

List of materials used in this article

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