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Summary

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Abstract

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of "contaminating" progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

Introduction

Monocytes, neutrophils, and dendritic cells (DCs) are myeloid cells that arise from hematopoietic progenitors, primarily in the bone marrow, by a process called myelopoiesis. Common myeloid progenitors (CMPs) have the potential to produce myeloid cells, as well as megakaryocytes and erythrocytes, but not lymphoid cells. Granulocyte-monocyte progenitors (GMPs), which are derived from CMPs, produce granulocytes and monocytes, but have lost megakaryocyte and erythrocyte potential. Monocytes and classical and plasmacytoid DCs (cDCs/pDCs) are also thought to arise from common progenitors known as monocyte-DC progenitors (MDPs), which are produced by CMPs. Gradual restriction of lineage potential ultimately results in lineage-committed progenitors: granulocyte progenitors, monocyte progenitors, and dendritic cell progenitors (Figure 1).

Weissman and colleagues reported that CMPs are found in the Lin^– c-Kit⁺ Sca-1^– (LKS^–) CD34⁺ FcγR^lo fraction of mouse bone marrow, while GMPs are contained in the LKS^– CD34⁺ FcγR^hi fraction¹. However, these "CMP" and "GMP" fractions are very heterogeneous. For instance, the "GMP" fraction also contains lineage-committed granulocyte progenitors and monocyte progenitors^1,2. MDPs were separately reported to be CX3CR1⁺ Flt3⁺ CD115⁺ progenitors that also express CD34 and FcγR^3,4. MDPs give rise to cDC/pDC-producing common DC progenitors (CDPs), which have been reported to express lower levels of c-Kit (CD117) and are not included in the LKS^– fraction⁵.

It was previously assumed that monocytes arise via a single pathway (CMP-GMP-MDP-monocyte). Consistent with this model, monocyte-committed progenitors produced by GMPs (named monocyte progenitors, MPs)² and MDPs (named common monocyte progenitors, cMoPs)⁶ appear to be the same cells on the basis of shared surface marker expression. However, we recently demonstrated that monocytes are produced independently by GMPs and MDPs, and were able to distinguish between MPs and cMoPs by single-cell RNA sequencing⁷.

We recently modified the Weissman "CMP" and "GMP" gating strategy to identify 6 subfractions of C57BL/6J mouse bone marrow containing different oligopotent and lineage-committed myeloid progenitor subsets. We first reported that staining for Ly6C and CD115 permits the isolation of oligopotent GMPs, as well as granulocyte progenitors (GPs) and monocyte progenitors (both MPs and cMoPs, which we are currently unable to separate) from the "GMP" fraction² (LKS^– CD34⁺ FcγR^hi gate; Figure 1). We subsequently demonstrated that MDPs are predominantly found in the "CMP" fraction (LKS^– CD34⁺ FcγR^lo gate), which also contains Flt3⁺ CD115^lo and Flt3^– subsets⁷ (Figure 1). The CMP-Flt3⁺ CD115^lo fraction yields both GMPs and MDPs upon adoptive transfer. The CMP-Flt3^– subset contains progenitors that appear to be intermediates between CMP-Flt3⁺ CD115^lo cells and GMPs. Unlike MDPs, both the CMP-Flt3⁺ CD115^lo and CMP-Flt3^– fractions also possess megakaryocyte and erythrocyte potential.

It is important to note, however, that it is currently unclear whether the "CMP" fractions contain progenitors that are truly oligopotent (e.g., individual cells within the CMP-Flt3⁺ CD115^lo fraction that possess neutrophil, monocyte, DC, megakaryocyte, and erythrocyte potential), or alternatively, comprise a mixture of progenitors with more restricted lineage potential. Colony forming assays (methylcellulose cultures) revealed cells with granulocyte (neutrophil), erythrocyte, monocyte and megakaryocyte potential (GEMM cells) in the "CMP", CMP-Flt3⁺ CD115^lo and CMP-Flt3^– fractions^1,7, but do not permit the assessment of DC potential. In contrast, colony forming assays demonstrated the existence of oligopotent GMPs (progenitors with both neutrophil and monocyte potential) in the "GMP" fraction^1,2, and this is supported by recent single-cell transcriptomic analysis⁸. It is not currently known, however, whether these oligopotent GMPs also produce other granulocytes (eosinophils, basophils, and mast cells).

Based on these studies, we now demonstrate how 7 surface markers (c-Kit, Sca-1, CD34, FcγR, Flt3, Ly6C and CD115) can be used to identify and isolate these 6 subsets of oligopotent and lineage-committed myeloid progenitors. The protocol described here can be applied for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments in mice, and molecular analysis (bulk and single-cell RNA sequencing, Western blotting, etc.).

The protocol consists of 3 stages: 1) preparation of a single cell suspension of bone marrow cells, 2) enrichment for hematopoietic progenitors (magnetic-activated cell sorting), and 3) identification, and isolation if desired, of progenitor subsets by flow cytometry (using an analyzer or a sorter, as appropriate). The first step is the isolation of bone marrow cells from the femurs and tibias of euthanized mice and is similar to other previously described protocols⁹. Next, the sample is enriched for stem and progenitor cells using a cocktail of antibodies against cell surface markers of erythrocytes, neutrophils, monocytes, lymphocytes, etc., to deplete the differentiated cells. This is not mandatory, but strongly recommended to optimize detection of the progenitor subsets, and to reduce the quantity of antibodies needed for progenitor identification and the time required for flow cytometry. The lineage depletion protocol below describes Magnetic-Activated Cell Sorting (MACS) using a Mouse Lineage Cell Depletion Kit (which contains biotinylated antibodies against CD5, CD45R (B220), CD11b, Gr-1 (Ly6G/C), 7-4, and Ter-119, plus anti-biotin microbeads) and an automated magnetic separator. The final step is the identification (and sorting, if desired) of the progenitor subsets by flow cytometry. The antibody panel described below (see also Table 1) has been designed to be used in a flow cytometer (analyzer or sorter) with 4 lasers (405 nm, 488 nm, 561 nm, 640 nm).

Figure 1: Neutrophil, monocyte and DC progenitors and differentiation pathways. The recently revised model of myelopoiesis⁷ is illustrated, with the Weissman gates for "CMPs" (blue) and "GMPs" (green)¹ overlaid. This figure has been modified from Yáñez et al. 2017⁷. Please click here to view a larger version of this figure.

Protocol

All methods described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Cedars-Sinai Medical Center. 1. Isolation of Mouse Bone Marrow and Preparation of a Single Cell Suspension Euthanize the mouse in accordance with institutional guidelines. Place the euthanized mouse on its back and spray it with 70% ethanol (EtOH). Make a small (3 – 5 mm) incision in each hind limb at the ankle level using sharp, pointed scissors, and pull the skin up towards the body to remove it from the legs and expose the muscles and bones. Remove the muscles (quadriceps, hamstrings, etc.) from the bones by trimming them with scissors, following the direction of the bone. Be careful not to damage the bones. Dislocate the femurs from the hip joints and cut away the connective tissue and muscle to detach the legs, being careful not to cut into the bones. Remove the feet by dislocating the ankles from the tibias, place the bones in a tube of sterile PBS or media, and transport them on ice to the tissue culture room. NOTE: For some other applications (e.g., deriving macrophages from total bone marrow cells) it is possible to leave the bones on ice for a while, but for progenitor isolation it is important to proceed as quickly as possible to avoid downregulation of important surface markers (especially CD115). In a biosafety cabinet, remove any remaining soft tissue from the bones by rubbing them with tissue paper. Note: The protocol should be performed in a biosafety cabinet if a sterile sample is required for cell culture or in vivo assays after FACS sorting. If sterile cells are not required, the whole process can be performed on the lab bench. Briefly immerse the bones in 70% EtOH to disinfect them, and then in sterile PBS to wash them. Separate the femurs from the tibias and fibulas by cutting through the knee, and discard the fibulas. Cut the ends off of the bones with sharp scissors. Harvest bone marrow with cold sterile PBS as follows until the bones appear white: Grip each bone with forceps, and insert a 26G needle connected to a 10 mL syringe containing cold sterile PBS into the bone shaft at one end. Hold the bone above a 50 mL centrifuge tube and flush 2 – 5 mL PBS through the bone to harvest the marrow. Harvest the marrow from all 4 bones per mouse (2 femurs and 2 tibias). Gently pipette up and down with a 1 mL pipette to disaggregate the bone marrow to form a cell suspension. Filter the bone marrow suspension through a piece of 30 μm nylon mesh to eliminate bone pieces and cell aggregates, thereby generating a single cell suspension. Centrifuge the single cell suspension at 280 x g for 5 min, and resuspend the cell pellet in sterile staining buffer (PBS + 0.5% FBS + 2 mM EDTA), using 5 mL staining buffer per mouse (e.g., 10 mL for cells pooled from the femurs and tibias of 2 mice). Take a 10 μL aliquot of the sample, mix it with 10 μL Trypan Blue, and load 10 μL of the resulting Trypan Blue-labeled sample on to a hemocytometer. Count the cells using a light microscope. NOTE: If the cells are too concentrated for reliable counting, dilute the sample prior to adding Trypan Blue. 2. Progenitor Enrichment by Magnetic-Activated Cell Sorting (MACS) Centrifuge the entire single cell suspension (or as many cells as are required to obtain the desired yield) at 280 x g for 5 min at 4°C and resuspend the cell pellet in 40 μL staining buffer (PBS + 0.5% FBS + 2 mM EDTA) per 107 cells. NOTE: It is also possible to use MACS buffer from the lineage depletion kit manufacturer (see Table of Materials) as an alternative to the staining buffer. Perform the lineage depletion according to the instructions supplied with the lineage depletion kit. The following instructions are for the kit seen in the Table of Materials. If a different kit is used, follow the manufacturer's instructions and then proceed to step 2.3. Add 10 μL biotin-antibody cocktail per 107 cells, mix by pipetting and incubate for 10 min at 4 °C. Add 30 μL staining buffer/MACS buffer per 107 cells and mix. Add 20 μL anti-biotin microbeads per 107 cells. Mix well and incubate for an additional 15 min at 4 °C. NOTE: Vortex the microbeads before adding them to the cells to make sure that they are completely dispersed. Add 1 mL staining buffer/MACS buffer per 107 cells, centrifuge the cells at 280 x g for 5 min, and resuspend the cell pellet in 500 μL staining buffer/MACS buffer for up to 108 cells. For more than 108 cells, scale up the volume of staining buffer/MACS buffer accordingly. Prepare the automated magnetic separator according to the manufacturer's instructions. Place the tube containing the labeled cells into the separator and choose the negative selection program. Collect the negative fraction, which contains the progenitor-enriched lineage-negative (Lin-) cells. Discard the positive fraction, which contains the magnetically-labeled lineage-positive cells. Centrifuge the Lin- cells at 280 x g for 5 min, and resuspend the cell pellet in 2 mL staining buffer/MACS buffer per mouse (e.g., 4 mL if bone marrow was pooled from 2 mice). Note: The pellet should now be white instead of red because the erythrocytes have been depleted. Take a 10 μL aliquot of the Lin- cells, mix it with 10 μL Trypan Blue, and load 10 μL of the resulting Trypan Blue-labeled sample on to a hemocytometer. Count the cells using a light microscope. NOTE: If the cells are too concentrated for reliable counting, dilute the sample prior to adding Trypan Blue. 3. Myeloid Progenitor Identification and Isolation by Fluorescence-activated Cell Sorting (FACS) Label 9 microcentrifuge (1.5 or 2 mL) tubes for: 1) unstained cells, 2-8) cells single stained with fluorophore-conjugated antibodies against FcγR (CD16/32), c-Kit, Sca-1, CD34, Flt3 (CD135), Ly6C and CD115 for voltage selection and color compensation, and 9) sample cells to be stained with all 7 antibodies for progenitor identification and sorting. Distribute 100,000 cells to each of the control tubes (tubes 1 – 8), and all the remaining cells (or fewer if desired) to the sample tube (tube 9). NOTE: If the sample volume is greater than 1.5 – 2 mL, it may be necessary to distribute the sample cells into a 15 mL tube, centrifuge them and resuspend the pellet (step 3.3 below), and then transfer the cells to a microcentrifuge tube for the subsequent staining steps. Centrifuge the cells at 1,500 x g for 5 min, and resuspend the control tube pellets in 100 μL staining buffer (PBS + 0.5% FBS + 2 mM EDTA) and the sample tube pellet in 100 μL staining buffer per 5 x 106 cells (e.g., 200 μL for 1 x 107 cells). Add 2 μg/mL anti-CD16/CD32-APC-Cy7 to the FcγR single stain tube and the sample tube. Vortex gently to mix, and incubate for 10 min at 4 °C. NOTE: It is important to stain the sample cells with the FcγR (CD16/32) antibody prior to adding the other antibodies to prevent competition due to non-specific Fc-mediated antibody binding. Do not incubate the cells with an FcγR blocking reagent. Add the other antibodies to the corresponding single stain tubes (1 antibody each) and the sample tube (all 6 antibodies): 10 μg/mL c-Kit-Pacific Blue, 2 μg/mL Sca-1-PE-Cy7, 25 μg/mL CD34-FITC, 10 μg/mL Flt3-APC, 2 μg/mL Ly6C-PerCP-Cy5.5, and 4 μg/mL CD115-PE. Vortex gently, and incubate for 15 min at 4 °C. Add 900 μL staining buffer to the control tubes, and 1 mL staining buffer per 107 cells to the sample tube. Centrifuge the cells at 1,500 x g for 5 min and resuspend the cell pellet in staining buffer: 200 μL per control tube, and 500 μL per 25 x 106 cells in the sample tube. Transfer the resuspended cells to 5 mL FACS tubes. NOTE: If the sample volume is greater than 1.5 – 2 mL, it may be necessary to transfer the cells to a 15 mL tube for centrifugation. Prepare the flow cytometer (analyzer or sorter) according to the manufacturer's instructions. Run the unstained and single stained control cells through the flow cytometer to set up the voltages and color compensations. Run the sample cells, gate the progenitor subsets as outlined below and summarized in Figure 2, and if desired, perform FACS sorting to isolate the relevant progenitor subsets. NOTE: It is typically necessary to acquire approximately 200,000 cells to obtain at least 1,000 events in each progenitor gate. Create a dot plot of all cells, displaying FSC-A (x-axis) and SSC-A (y-axis), and gate to exclude debris and dead cells => "live" cells. Create a dot plot of "live" cells, displaying FSC-H (x-axis) and FSC-W (y-axis), and gate to exclude doublets => live/singlet (FSC) gate. Create a dot plot of live/singlet (FSC) cells, displaying SSC-H (x-axis) and SSC-W (y-axis), and gate to exclude doublets => live/singlet (FSC)/singlet (SSC) gate. Create a dot plot of live/singlet (FSC)/singlet (SSC) cells, displaying Sca-1 (x-axis) and c-Kit (y-axis), and gate to select c-Kit+ Sca-1- cells => LKS- cells. Create a dot plot of LKS- cells, displaying CD34 (x-axis) and FcγR (y-axis), and gate to select CD34+ FcγRlo cells => "CMPs", and CD34+ FcγRhi cells => "GMPs". Note: It’s important to gate the “CMPs” and “GMPs” as precisely as possible. It’s helpful to use a pseudocolor density plot or a contour plot for accurate gating. Create a dot plot of "CMPs", displaying CD115 (x-axis) and Flt3 (y-axis), and gate to select: Flt3+ CD115lo cells => CMP-Flt3+ CD115lo cells, Flt3- CD115lo cells => CMP-Flt3- cells, Flt3+ CD115hi cells => MDPs. Create a dot plot of "GMPs", displaying Ly6C (x-axis) and FcγR (y-axis), and gate to select Ly6C-cells => "GMP-Ly6C- cells", and Ly6C+ cells => "GMP-Ly6C+ cells". Create a dot plot of "GMP-Ly6C- cells", displaying CD115 (x-axis) and Flt3 (y-axis), and gate to select: Flt3- CD115lo cells => GMPs. Create a dot plot of "GMP-Ly6C+ cells", displaying CD115 (x-axis) and Flt3 (y-axis), and gate to select: Flt3- CD115lo cells => GPs, Flt3- CD115hi cells => MPs+cMoPs.

Representative Results

Using the protocol described above, it is possible to obtain ~100 million cells (including red blood cells, or ~50 million nucleated cells) from both femurs and tibias (2 legs) of one C57BL/6J mouse (6 – 8 weeks old, male or female). 1 – 2 million Lin- cells can be isolated per mouse by MACS depletion of Lin+ cells. Each of the 6 myeloid progenitor subsets constitutes ~1 – 4% of the Lin- cells. L…

Discussion

The Weissman gating strategy for mouse myeloid progenitor identification¹ has been the gold standard for immunologists and hematologists for nearly 20 years, but it is now apparent that the "CMP" and "GMP" gates are very heterogeneous and more precise gating strategies are needed. The protocol that we have described here permits the identification of oligopotent and lineage-committed subsets in C57BL/6J mice for more precise quantification of specific myeloid progenitors and mappin…

Materials

Mouse: Wild-type C57BL/6J (CD45.2)	The Jackson Laboratories	Cat#JAX:000664
Lineage Cell Depletion Kit, mouse	Miltenyi Biotec	Cat#130-090-858
Rat anti-mouse CD34 (clone RAM34) FITC	BD Biosciences	Cat#553733
Rat anti-mouse CD16/CD32 (FcγR; clone 93) APC-Cy7	BioLegend	Cat#101327
Rat anti-mouse Ly6A/E (Sca-1; clone 108113) PE-Cy7	BioLegend	Cat#108114
Rat anti-mouse CD117 (c-Kit; clone 2B8) Pacific Blue	BioLegend	Cat#105820
Rat anti-mouse Ly6C (clone HK1.4) PerCP-Cy5.5	BioLegend	Cat#128012
Rat anti-mouse CD115 (clone AFS98) PE	BioLegend	Cat#135506
Rat anti-mouse CD135 (Flt3; clone A2F10.1) APC	BD Biosciences	Cat#560718
CountBright Absolute Counting Beads	Thermo Fisher Scientific	Cat#C36950
AutoMACS Separator	Miltenyi Biotec	N/A	Use the "deplete" program
BD LSRFortessa	BD Biosciences	N/A	5 lasers, 15 colors
BD FACS Aria III cell sorter	BD Biosciences	N/A	5 lasers, 13 colors
FlowJo	FlowJo, LLC	https://www.flowjo.com	For further analysis of the .fcs files