Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

Summary

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

Abstract

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Introduction

Flow cytometry is an invaluable method to characterize and prospectively isolate immune and hematopoietic cells. It is also increasingly being used to analyze stromal and epithelial populations of different tissues. The hematopoietic stem cell (HSC) has unique properties of self-renewal and multipotency. In adult mammals, HSCs primarily reside in the bone marrow (BM), where they receive quiescence and survival signals from the surrounding microenvironment or niche¹. HSCs are formally defined according to functional assays². Nevertheless, several landmark papers have shown the usefulness of flow cytometry to identify HSCs. Through the use of limited cell surface markers, it is possible to discriminate hematopoietic populations that are highly enriched in HSCs³. Flow cytometry is, therefore, a central method in the stem cell field. It has been extensively used to evaluate the impact of putative niche cell types and niche factors on HSCs. By combining flow cytometry with imaging and functional assays, it has been shown that HSCs are critically supported by perivascular mesenchymal stem cells (MSCs) and endothelial cells (ECs). BM MSCs are a heterogenous group and have different cytokine contributions⁴, but it is well established that leptin receptor (LepR)⁺ MSCs are key niche cells¹. BM ECs are also highly heterogeneous and can be part of sinusoids, arterioles, and type H/transitional vessels⁵. Different studies have shown the nuanced contribution of these different ECs. For example, endosteal sinusoidal ECs are spatially closer to quiescent HSCs⁶, while non-migratory HSCs with lower levels of reactive oxygen species are located near arteriolar ECs⁷. The endosteal versus central location of niches is also very important. Endosteal type H vessels are associated with perivascular stromal cells that are lost with aging, leading to the loss of HSCs⁸. In acute myeloid leukemia, central ECs are expanded, while endosteal vessels and endosteal HSCs are lost⁹.

Most studies in the field have focused on hematopoiesis itself and on the cell's extrinsic regulation of HSCs. It has been, however, increasingly recognized that there is a need to better characterize the niches that regulate other progenitors, namely multipotent progenitors (MPPs), particularly considering that they are the main drivers of hematopoiesis in steady state¹⁰. In contrast with a fixed hierarchical structure, recent studies have shown that hematopoiesis is a continuum in which HSCs differentiate into biased MPPs at an early stage¹¹. MPPs have been named after different classification schemes¹², but a recent consensus paper by the international society for experimental hematology (ISEH) proposed MPPs to be discriminated as early MPPs and according to their lymphoid (MPP-Ly), megakaryocytic and erythroid (MPP-Mk/E), and myeloid (MPP-G/M) bias¹³. The use of flow cytometry will be critical in further studying the importance of BM niches in the regulation of these populations. Current flow cytometry methods use variable gating strategies to differentiate HSPCs and identify stromal cells, namely ECs, using inconsistent markers. The goal of the current method is to present a simple and reproducible workflow of BM staining to identify HSPC subpopulations, heterogeneous groups of ECs, and LepR⁺ MSCs. We believe this technique, although comparable with previously reported methods (see, for example, reference¹⁴), provides an updated and easy-to-implement protocol for the phenotypic analysis of hematopoietic cells in the two functional marrow areas, endosteal and central BM^8,15, as well as BM stromal niche cells.

Protocol

The animals used in this protocol were housed at the i3S animal facility under specific pathogen-free conditions in a 12 h light-dark cycle and temperature-controlled environment. Free access to standard rodent chow and water was provided. All the animals received humane care according to the criteria outlined by the Federation of European Laboratory Animal Science Associations for the care and handling of laboratory animals (EU Directive 2010/63/EU). The experimental procedure performed on the animals (euthanasia) was approved by the i3S Animal Ethics Committee (ref. DD_2019_15) and the Direção-Geral de Alimentação e Veterinária. Details of the materials used throughout this protocol can be found in the Table of Materials.

1. Preparation of solutions and staining cocktails

1. Phosphate-buffered saline (PBS): Dissolve five PBS tablets into 1 L of distilled water. Store at room temperature (RT).
2. PBS 2% fetal bovine serum (FBS): Add 10 mL of FBS to 500 mL of PBS. Store at 4 °C.
3. Red blood cell (RBC) lysis buffer (1x): Add 50 mL of 10x RBC lysis buffer to 450 mL of deionized water. Store at 4 °C.
4. Collagenase IV and dispase II solution: Dissolve 30 mg of collagenase IV and 60 mg of dispase II in 30 mL of HBSS for a 1 mg/mL collagenase IV and 2 mg/mL dispase II solution. Prepare fresh before use.
5. Fc receptors blocking solution: Add 10 µL of purified anti-mouse CD16/32 antibody to 490 µL of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 °C until use.
6. Fluorescent cell viability dye staining solution: Add 2 µL of fluorescent cell viability dye to 1 mL of PBS. Prepare fresh before use.
7. Biotin lineage cocktail: Mix 100 µL of each of the following antibodies: biotin anti-mouse CD3ε, biotin anti-mouse CD4, biotin anti-mouse CD8a, biotin anti-mouse/human CD11b, biotin anti-mouse/human CD45R/B220, biotin anti-mouse Ly-6G/Ly-6C (Gr-1), and biotin anti-mouse TER-119/erythroid cells. Store at 4 °C until use.
8. HSCs primary staining cocktail: Add 10 µL of biotin lineage cocktail, 10 µL of immunofluorescently tagged 510 anti-mouse CD150 (SLAM), 10 µL of phycoerythrin (PE) anti-mouse Flk2 (CD135), 10 µL of peridinin-chlorophyll-protein (PerCP) anti-mouse Ly-6A/E (Sca-1), 10 µL of PE/Cyanine7 anti-mouse CD48, and 10 µL of allophycocyanin (APC)/Cyanine7 anti-mouse CD117 (c-kit) to 940 µL of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 °C protected from light until use.
9. Stromal primary staining cocktail: Add 5 µL of mouse leptin R biotinylated antibody, 6.7 µL of PE anti-mouse endomucin antibody, 10 µL of PerCP anti-mouse Ly-6A/E (Sca-1), 4 µL of PE/Cyanine7 anti-mouse CD31, 10 µL of APC/Cyanine7 anti-mouse CD45, and 10 µL of APC/Cyanine7 anti-mouse TER-119/erythroid cells to 954 µL of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 °C protected from light until use.
10. HSCs/stromal secondary staining solution: Add 1 µL of APC streptavidin to 1 mL of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 °C protected from light until use.
11. ECs staining cocktail: Add 10 µL of PE anti-mouse endomucin antibody, 10 µL of PerCP anti-mouse Ly-6A/E (Sca-1), 10 µL of PE/Cyanine7 anti-mouse CD31, 10 µL of Alexa Fluor 647 anti-mouse CD54/ICAM-1, 10 µL of APC/Cyanine7 anti-mouse CD45, and 10 µL of APC/Cyanine7 anti-mouse TER-119/erythroid cells to 940 µL of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 °C protected from light until use.
12. DAPI staining solution: Add 1 drop of DAPI reagent to 500 µL of PBS. Prepare fresh before use.

2. Sample extraction

1. Euthanize the animal by cervical dislocation (details of the animals used to produce the data presented in this study can be found in Supplementary Table 1). Place the animal with the belly up on a Petri dish and spray with 70% ethanol. Make a cut above the abdomen using scissors and pull away the skin all the way to the ankles.
2. Grab one leg and, using scissors, cut at its bottom (where the joint between the leg and the hip is) to separate it from the animal. This step will also serve as a secondary confirmatory method of euthanasia. Remove the foot from the leg by cutting at the ankle. Place the leg in ice-cold PBS 2% FBS. If required, repeat the step with the other leg.
3. Grab the hip bone and, using scissors cut behind it to separate it from the animal. Place the hip bone in ice-cold PBS 2% FBS. If required, repeat the step with the other hip bone.
4. Place the leg(s) and hip bone(s) in a Petri dish and, using a sterile scalpel, clean the bones. Separate the tibia and femur by cutting at the knee with the scalpel. Place the clean bones in ice-cold PBS 2% FBS.

3. Sample processing for analysis of hematopoietic cell populations in total bone marrow

1. Place a femur or tibia in a mortar with ice-cold PBS 2% FBS and crush the bone by gently pressing it against the wall of the mortar using the pestle. BM cells are released into the solution contained in the mortar.
2. Pipette the resulting cell suspension up and down using a 10 mL pipette to homogenize and transfer into a 50 mL tube through a 40 µm cell strainer placed on top of the tube.
3. If the crushed bones do not look white at this point, add more PBS 2% FBS to the mortar and repeat crushing, and then pipette up and down and transfer the solution into the same 50 mL tube through the same 40 µm cell strainer.
4. Rinse the mortar with some more PBS 2% FBS and transfer the solution into the same 50 mL tube through the same 40 µm cell strainer. Centrifuge the 50 mL tube at 500 x g for 5 min at 4 °C.
5. Discard the supernatant and resuspend the cell pellet in 5 mL of RBC lysis buffer (1x) at RT by pipetting up and down several times. Following a 2 min incubation at RT, add 15 mL of PBS 2% FBS.
6. Centrifuge the 50 mL tube at 500 x g for 5 min at 4 °C. If the cell pellet still looks reddish, go back to step 3.5. If not, discard the supernatant, resuspend the pellet in 200 µL of PBS, and transfer the cell suspension into a well of a 96-well V-bottom plate for staining.
7. Centrifuge the plate at 500 x g for 3 min at 4 °C. Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 200 µL of fluorescent dye staining solution. Incubate the plate for 15 min at RT in the dark.
8. Centrifuge plate at 500 x g for 3 min at 4 °C. Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 200 µL of PBS 2% FBS to wash.
9. Centrifuge the plate at 500 x g for 3 min at 4 °C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 µL of Fc receptors blocking solution. Incubate the plate for 10 min at 4 °C protected from light.
10. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 µL of HSCs primary staining cocktail. Incubate the plate for 15 min at RT in the dark.
11. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 µL of HSCs secondary staining solution. Incubate the plate for 15 min at RT in the dark.
12. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C and discard the supernatant by flipping the plate onto absorbent paper.
13. Resuspend the pellet in 250 µL of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 µm cell strainer. Keep on ice protected from light until flow cytometry analysis.
    ​NOTE: If interested in determining the absolute numbers of hematopoietic cell populations, add Calibrite Beads to the polystyrene tubes before analysis in the cytometer¹⁶.

4. Sample processing for the analysis of stromal cell populations in total bone marrow

1. Place a femur or tibia in a mortar with ice-cold PBS 2% FBS and crush the bone by gently pressing it against the wall of the mortar using the pestle. Cut the tip of a P1000 tip using scissors and use it to transfer all the content of the mortar into a 50 mL tube (do not pass through a cell strainer).
2. Centrifuge the 50 mL tube at 500 x g for 5 min at 4 °C, discard the supernatant, and resuspend the pellet in 3 mL of collagenase IV and dispase II solution. Incubate the tube at 37 °C for 40 min.
3. Top up the tube to 25 mL with PBS 2% FBS and vortex to mix. Transfer the content of the 50 mL tube to a new 50 mL tube through a 100 µm cell strainer. Add 15 mL of PBS 2% FBS to the old 50 mL tube and transfer the solution into the same new 50 mL tube through the same cell strainer. Centrifuge at 500 x g for 5 min at 4 °C.
4. Discard the supernatant and resuspend the cell pellet in 5 mL of RBC lysis buffer (1x) at RT by pipetting up and down several times. Following a 2 min incubation at RT, add 15 mL of PBS 2% FBS.
5. Centrifuge the 50 mL tube at 500 x g for 5 min at 4 °C. If the cell pellet still looks reddish, go back to step 4.4. If not, discard the supernatant, resuspend the pellet in 200 µL of PBS 2% FBS, and transfer the cell suspension into a well of a 96-well V-bottom plate for staining. Centrifuge the plate at 500 x g for 3 min at 4 °C.
6. If performing stromal staining, proceed to step 4.7. If performing EC staining, proceed to step 4.12.
7. Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 100 µL of stromal primary staining cocktail. Incubate the plate for 15 min at RT in the dark.
8. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 µL of stromal secondary staining solution. Incubate the plate for 15 min at RT in the dark.
9. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C. Discard the supernatant by flipping the plate onto absorbent paper.
10. Resuspend the pellet in 250 µL of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 µm cell strainer. Keep on ice protected from light.
11. Just before going to the cytometer, add 35 µL of DAPI staining solution to the tube containing the cell suspension for flow cytometry analysis and incubate the tube at RT for 5 min in the dark. Following this incubation, keep on ice protected from light until flow cytometry analysis.
12. Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 100 µL of ECs staining cocktail. Incubate the plate for 15 min at RT in the dark.
13. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C.
14. Discard the supernatant by flipping the plate onto absorbent paper. Resuspend the pellet in 250 µL of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 µm cell strainer. Keep on ice protected from light.
15. Just before going to the cytometer, add 35 µL of DAPI staining solution to the tube containing the cell suspension for flow cytometry analysis and incubate the tube at RT for 5 min in the dark. Following this incubation, keep on ice protected from light until flow cytometry analysis.
    ​NOTE: If interested in determining the absolute numbers of stromal and endothelial cell populations, add Calibrite Beads to the polystyrene tubes before analysis in the cytometer¹⁶.

5. Sample processing for the analysis of hematopoietic cell populations in crushed and flushed BM

1. Place a femur on a Petri dish and cut the ends of the bone using a sterile scalpel.
2. Flush the central part of the bone (diaphysis) by passing 100 µL of PBS 2% FBS through the inside of the bone once from each side using a no dead volume 0.5 mL insulin syringe and collecting the solution in a microcentrifuge tube containing 1 mL of PBS 2% FBS. Subsequently, transfer the cell suspension into a 50 mL tube labeled as flushed BM containing 4 mL of PBS 2% FBS. Keep on ice.
3. Place the ends of the bone in a mortar with ice-cold PBS 2% FBS and crush by gently pressing it against the mortar wall using the pestle. Pipette the resulting cell suspension up and down using a 10 mL pipette to homogenize and transfer into a 50 mL tube labeled as crushed BM through a 40 µm cell strainer.
4. If the crushed bone does not look white at this point, add more PBS 2% FBS to the mortar and repeat the crushing. Then pipette up and down and transfer the solution into the same 50 mL tube (crushed BM) through the same 40 µm cell strainer.
5. Rinse the mortar with some more PBS 2% FBS and transfer the solution into the same 50 mL tube (crushed BM) through the same 40 µm cell strainer.
6. Centrifuge the 50 mL tubes corresponding to the flushed and crushed samples at 500 x g for 3 min at 4 °C.
7. Follow steps 3.5 to 3.13 (section 3) with the flushed and crushed BM samples.

6. Preparation of single-color controls (SCCs) for flow cytometry analysis

1. Follow steps 3.1 to 3.6 (section 3) with an extra bone from one of the animals not used for the main analysis, transferring the final cell suspension into a microcentrifuge tube containing 1 mL of PBS 2% FBS instead of a well of a 96-well V-bottom plate.
2. Transfer 100 µL of the cell suspension to 8 wells of a 96-well V-bottom plate, 100 µL into a 5 mL polystyrene round-bottom tube containing 100 μL of PBS 2% FBS labeled as DAPI SCC, and the remaining cell suspension into a 5 mL polystyrene round-bottom tube containing 100 μL of PBS 2% FBS labeled as unstained. Keep the tubes on ice protected from light.
3. Centrifuge the plate at 500 x g for 3 min at 4 °C and discard the supernatants by flipping the plate onto absorbent paper.
4. Resuspend one pellet in 100 µL of fluorescent dye staining solution and the remaining ones in 100 µL of PBS 2% FBS.
5. Add 0.5 µL of the following antibodies, apart from the lineage cocktail of antibodies for which add 2 µL and the PECy7 CD31 antibody for which add 0.3 µL, to each of the wells with cell suspension in PBS 2% FBS (one antibody per well, same antibodies used in the main analysis panels or same fluorophore antibodies with similar fluorescence intensity and epitope abundance): biotin lineage cocktail, immunofluorescently tagged 510 anti-mouse CD150 (SLAM), PE anti-mouse Flk2 (CD135), PerCP anti-mouse Ly-6A/E (Sca-1), immunofluorescently tagged 647 anti-mouse CD54/ICAM-1, PE/Cyanine7 anti-mouse CD48, and APC/Cyanine7 anti-mouse CD117 (c-kit). Incubate the plate for 15 min at RT in the dark.
6. Add 100 µL of PBS 2% FBS to each well and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C and discard the supernatants by flipping the plate onto absorbent paper.
7. Resuspend all the pellets apart from the one corresponding to the cells incubated with the lineage cocktail of antibodies in 180 µL of PBS 2% FBS and transfer the cell suspensions into 5 mL polystyrene round-bottom tubes through 40 µm cell strainers. Keep on ice protected from light until flow cytometry analysis.
8. Resuspend the cells incubated with the lineage cocktail of antibodies in 100 µL of HSCs/stromal secondary staining solution. Incubate the plate for 15 min at RT in the dark.
9. Add 100 µL of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 °C.
10. Resuspend the pellet in 180 µL of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 µm cell strainer. Keep on ice protected from light until flow cytometry analysis.
11. Just before going to the cytometer, add 35 µL of DAPI staining solution to the DAPI SCC tube and incubate the tube at RT for 5 min in the dark. Following this incubation, keep on ice protected from light until flow cytometry analysis.

Results

Representative plots of flow cytometry analysis of HSCs and MPPs in a healthy young adult C57Bl/6 mouse are shown in Figure 1. The gating strategy follows the latest harmonizing nomenclature proposed by the ISEH¹³. When analyzing the impact of a perturbation, such as infection or cancer, it is important to use a control mouse as a reference for normal gates. Fluorescence-minus-one (FMO) controls can be particularly useful to delineate the boundaries of the gates, but it should be noted that blindly setting up gate limits based on FMOs may omit target cells or include non-target cells. For example, the lower the intensity of the CD48 set, the higher the enrichment for quiescent long-term HSCs¹⁷. Furthermore, different properties of certain populations rely on the intensity of specific markers. For example, HSCs expressing higher CD150 levels are more myeloid-biased¹⁸.

The frequencies and absolute numbers obtained in the analysis of hematopoietic cell populations applied to a total of four animals are presented in Table 1.

Figure 2 shows representative plots of flow cytometry analysis of ECs and MSCs. The majority of hematopoietic cells are excluded following a Ter119/CD45 negative selection. LepR⁺ MSCs can then be readily identified, while true ECs require a subsequent selection of Sca-1⁺ cells. While Sca-1 is often used in immunofluorescence studies to specifically mark arterioles, this is not the case in flow cytometry, as this technique is highly sensitive, and ECs always express a certain (even if low) degree of Sca-1 at the cell surface. By not selecting only Sca-1⁺ cells, other non-endothelial cells would be included in the analysis, such as CD31-expressing myeloid cells, which might significantly impact the quantification of ECs in the BM. ECs can be analyzed as a whole population or based on specific markers that partially reveal their heterogeneity. Endomucin in combination with CD31 is very useful to identify the functionally distinct type H endothelium¹⁵ (Figure 2). Furthermore, it has been previously shown that ICAM-1 expression allows the discrimination between arteriolar (aBMECs) and sinusoidal ECs (sBMECs) (Figure 3)²⁰.

The frequencies and absolute numbers obtained in the analysis of MSCs and endothelial cell populations applied to a total of four animals are presented in Table 2 and Table 3.

Although BM functional areas are not clearly delineated in evident histological regions, it is well established that bone-lining endosteal areas and central BM areas are enriched in certain cell types and events (e.g., the release of platelets/pro-platelets from megakaryocytes in the sinusoids of central BM areas). It is, therefore, useful to enrich for cell types in central and endosteal areas to separately analyze these two compartments. We applied a method of flushing the diaphysis to enrich for central cell types and crushing the metaphysis to enrich for endosteal cell types (Figure 4A). The quantification of aBMECs and sBMECs in flushed (central) and crushed (endosteal) BM tissue (Figure 4B) validates the applicability of this mechanical isolation method, as aBMECs are notoriously more abundant in endosteal regions, and sinusoids are more frequent in central BM areas.

Figure 1: Analysis of hematopoietic cell populations. Representative plots showing the gating strategy for the flow cytometry analysis of hematopoietic cell populations in total BM using the software provided. Abbreviations: FSC-H = forward scatter - height, FSC-A = forward scatter - area, Lin = lineage, LKS = Lin^- Sca-1⁺ c-Kit⁺ hematopoietic progenitor cells, MPPs^Ly = multipotent progenitors - lymphoid, MPPs^G/M = multipotent progenitors - granulocyte/macrophage, MPPs^Mk/E = multipotent progenitors - megakaryocyte/erythrocyte, MPPs = multipotent progenitors, HSCs = hematopoietic stem cells. Please click here to view a larger version of this figure.

Figure 2: Analysis of stromal cell populations. Representative plots showing the gating strategy for the flow cytometry analysis of stromal cells in total BM using the software provided. Abbreviations: FSC-H = forward scatter - height, FSC-A = forward scatter - area, MSCs = mesenchymal stem cells, LepR = leptin receptor, ECs = endothelial cells. Please click here to view a larger version of this figure.

Figure 3: Analysis of endothelial cells. Representative plots showing the gating strategy for the flow cytometry analysis of ECs in total BM using the software provided. Abbreviations: FSC-H = forward scatter - height, FSC-A = forward scatter - area, ECs = endothelial cells, aBMECs = arterial bone marrow endothelial cells, sBMECs = sinusoidal bone marrow endothelial cells. Please click here to view a larger version of this figure.

Figure 4: Analysis of EC populations in central and endosteal areas. (A) Representation of the different parts of a murine long bone. (B) Plots showing the statistically significant differences in the frequencies of CD31+ endothelial cells in aBMECs (enriched in the crushed sample) and sBMECs (enriched in the flushed sample). Each dot represents a mouse (n = 9), and samples are paired. The statistical test used was the paired T-test. **** denotes two-tailed P-value < 0.0001. Please click here to view a larger version of this figure.

Cell population	Mean of freq. of BM live cells ± SD (n=4) in total BM	Mean of cell count ± SD (n=4) per femur in total BM	Mean of freq. of BM live cells ± SD (n=4) in endosteal BM	Mean of freq. of BM live cells ± SD (n=4) in central BM
Lin-	3.05 ± 0.13	337610 ± 59414	3.56 ± 0.21	3.53 ± 0.19
LKS	0.096 ± 0.026	10206 ± 1794	0.102 ± 0.025	0.133 ± 0.022
MPPsLy	0.058 ± 0.011	6141 ± 716	0.058 ± 0.011	0.085 ± 0.007
MPPsG/M	0.011 ± 0.004	1233 ± 326	0.013 ± 0.003	0.014 ± 0.003
MPPsMk/E	0.0010 ± 0.0002	113 ± 21	0.0014 ± 0.0004	0.0012 ± 0.0005
MPPs	0.0092 ± 0.0045	940 ± 374	0.0105 ± 0.0048	0.0118 ± 0.0061
HSCs	0.0054 ± 0.0023	572 ± 191	0.0055 ± 0.0023	0.0059 ± 0.0016

Table 1: Quantitative data for hematopoietic cell populations. Mean of frequency in BM live cells of the different hematopoietic cell populations analyzed in total, endosteal, and central BM and cell counts per femur in total BM. Data shown as mean ± standard deviation (SD), n = 4.

Cell population	Mean of freq. of BM live cells ± SD (n=4)	Mean of cell count ± SD (n=4) per tibia
ECs	0.080 ± 0.011	4523 ± 1521
Type H ECs	0.041 ± 0.006	2299 ± 752
Type L ECs	0.038 ± 0.005	2103 ± 736
MSCs	0.180 ± 0.048	10270 ± 4282

Table 2: Quantitative data for stromal cell populations. Mean of frequency in BM live cells and cell counts per tibia for the different stromal cell populations analyzed in total BM. Data shown as mean ± standard deviation (SD), n = 4.

Cell population	Mean of freq. of BM live cells ± SD (n=4)	Mean of cell count ± SD (n=4) per tibia
ECs	0.064 ± 0.006	2255 ± 150
aBMECs	0.041 ± 0.010	1419 ± 286
sBMECs	0.023 ± 0.006	819 ± 250

Table 3: Quantitative data for endothelial cell populations. Mean of frequency in BM live cells and cell counts per tibia for the different endothelial cell populations analyzed in total BM. Data shown as mean ± standard deviation (SD), n = 4.

Supplementary Table 1: Details of the animals used in the study. Strain, sex, age, and weight of the animals used to produce the data shown in Figure 1, Figure 2, and Figure 3 and Table 1, Table 2, and Table 3. Abbreviation: g = grams. Please click here to download this File.

Discussion

While the protocol described is simple and easy to perform, special attention should be brought to specific steps. For example, when obtaining flushed BM (step 5.2), the volume or number of times indicated to pass PBS 2% FBS through the inside of the central part of the bone should not be exceeded, as this might result in significant contamination of the flushed sample by endosteal cell populations.

Alterations to the protocol can be made to facilitate its execution by the investigator. In sample extraction (section 2), legs and/or bones can be stored in PBS 2% FBS at 4 °C for up to 24 h before proceeding to sample processing and analysis. However, this time should be minimized when possible. During incubation with HSCs primary staining cocktail (step 3.10) and secondary staining cocktail (step 3.11), while a 15 min incubation at RT is indicated, this can be exchanged for a 30 min incubation at 4 °C.

The same applies to incubation with stromal primary staining cocktail (step 4.7) , stromal secondary staining solution (step 4.8), ECs staining cocktail (step 4.12) and incubation with antibodies for SCCs (step 6.5); while a 15 min incubation at RT is indicated, this can be exchanged for a 30 min incubation at 4 °C.

Another protocol for BM isolation by centrifugation of murine long bones was recently described²¹. While this protocol presents the advantage of faster isolation of the BM cells, the protocol described here allows for the analysis of cell populations residing in different areas of the bones.

A particular limitation of the current method is that it is solely based on phenotypic analysis by flow cytometry. This is particularly relevant in the case of HSCs, which would require further functional validation. This method can, however, be combined with the sorting of enriched populations and the study of these cells in long-term reconstitution assays and in vitro colony assays.

Regarding stromal cell analysis, in particular MSCs and ECs, it has been previously shown that, despite the optimization of BM processing for flow cytometry analysis, a significant number of cells are not isolated from the tissue and there is a suboptimal quantification of these cells when compared with other methods such as whole-mount imaging¹⁹. Nevertheless, flow cytometry is extremely useful to perform the quantitative and qualitative analysis of BM ECs and MSCs and to prospectively isolate them for functional and expression assays.

Another limitation of the current method is the use of a mechanical technique to separate the endosteal and central fractions, which are to some extent inevitably contaminated by cells from the other compartment. Nevertheless, the quantification of aBMECs and sBMECs as explained in the representative results section shows that the mechanical isolation is a robust method to enrich for cell populations in these areas.

The described method allows for the study of hematopoiesis in different settings and stromal niches of HSPCs. The phenotypic analysis here presented may be useful to study HSPC niches when combined with specific mouse mutants, namely EC and MSC Cre lines that enable the selective manipulation of gene expression in these populations. For example, the Cdh5(PAC)-CreERT2²² and the LepR-Cre²³ lines can be used to induce the expression of certain genes selectively in ECs and MSCs, respectively. The method is useful to study their impact on the stromal compartment as well as on HSPCs. Available Cre lines to study the vascular BM niche have been recently reviewed in Mosteo et al.⁵. The prospective study of LT-HSCs without transplantation has been limited by the lack of robust reporter lines. The recently described Mds1^GFP/+Flt3^{Cre 6} has been, however, shown to achieve high enrichment of LT-HSCs and good  discrimination from downstream progenitors expressing Flt3²⁴. The combination of this mouse line and other hematopoietic strains, such as Vav-iCre²⁵, with flow cytometry will allow the study of HSPCs and their relationship with the niche. Hematological malignancies are frequently associated with the disruption of hematopoiesis from the earliest stages, which is reflected in the alteration of the frequencies of hematopoietic stem and progenitor cells and of stromal populations⁹ that we have here presented for healthy C57Bl/6. Future studies should explore the application of similar methods to the one here described using new equipment based on spectral flow cytometry that enables a more extensive phenotypic characterization through the analysis of more markers (above 30 simultaneously).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 647 anti-mouse CD54/ICAM-1 antibody	BioLegend	116114
APC Streptavidin	BioLegend	405207
APC/Cyanine7 anti-mouse CD117 (c-kit) antibody	BioLegend	105826
APC/Cyanine7 anti-mouse CD45 antibody	BioLegend	103116
APC/Cyanine7 anti-mouse TER-119/erythroid cells antibody	BioLegend	116223
Biotin anti-mouse CD3ε antibody	BioLegend	100304
Biotin anti-mouse CD4 antibody	BioLegend	100404
Biotin anti-mouse CD8a antibody	BioLegend	100704
Biotin anti-mouse Ly-6G/Ly-6C (Gr-1) antibody	BioLegend	108404
Biotin anti-mouse TER-119/erythroid cells antibody	BioLegend	116204
Biotin anti-mouse/human CD11b antibody	BioLegend	101204
Biotin anti-mouse/human CD45R/B220 antibody	BioLegend	103204
Brilliant Violet 510 anti-mouse CD150 (SLAM) antibody	BioLegend	115929
Calibrite 2 Color Beads	BD Biosciences	349502
Collagenase IV	Merck Life Science	C1889
Dispase II	Merck Life Science	D4693
Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin	ThermoFisher Scientific	10500064
Hanks' Balanced Salt Solution (HBSS)	ThermoFisher Scientific	14175095
Mouse Leptin R Biotinylated Antibody	R&D systems	BAF497
NucBlue Fixed Cell Reagent (DAPI)	ThermoFisher Scientific	R37606	DAPI reagent
PE anti-mouse endomucin antibody	ThermoFisher Scientific	12-5851-82
PE anti-mouse Flk2 (CD135)	ThermoFisher Scientific	12-1351-82
PE/Cyanine7 anti-mouse CD31 antibody	BioLegend	102524
PE/Cyanine7 anti-mouse CD48 antibody	BioLegend	103424
PerCP anti-mouse Ly-6A/E (Sca-1) antibody	BioLegend	108122
Phosphate-buffered saline (PBS) tablets	Merck Life Science	P4417
Purified anti-mouse CD16/32 antibody	BioLegend	101302
RBC lysis buffer 10x	BioLegend	420302
Zombie Violet Fixable Viability Dye	BioLegend	423114	fluorescent dye