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.
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.
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+ FcγRlo fraction of mouse bone marrow, while GMPs are contained in the LKS– CD34+ FcγRhi fraction1. However, these "CMP" and "GMP" fractions are very heterogeneous. For instance, the "GMP" fraction also contains lineage-committed granulocyte progenitors and monocyte progenitors1,2. MDPs were separately reported to be CX3CR1+ Flt3+ CD115+ progenitors that also express CD34 and FcγR3,4. 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+ FcγRhi gate; Figure 1). We subsequently demonstrated that MDPs are predominantly found in the "CMP" fraction (LKS– CD34+ FcγRlo gate), which also contains Flt3+ CD115lo and Flt3– subsets7 (Figure 1). The CMP-Flt3+ CD115lo fraction yields both GMPs and MDPs upon adoptive transfer. The CMP-Flt3– subset contains progenitors that appear to be intermediates between CMP-Flt3+ CD115lo cells and GMPs. Unlike MDPs, both the CMP-Flt3+ CD115lo 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+ CD115lo 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+ CD115lo and CMP-Flt3– fractions1,7, 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" fraction1,2, 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.
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
2. Progenitor Enrichment by Magnetic-Activated Cell Sorting (MACS)
3. Myeloid Progenitor Identification and Isolation by Fluorescence-activated Cell Sorting (FACS)
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. Lineage depletion is efficient at depleting differentiated cells and enriching for progenitors, but the Lin– fraction contains a large proportion of c-Kit– cells in addition to c-Kit+ progenitors (Figure 3A). Progenitor yields after FACS sorting can vary depending on the sorter settings used (e.g., maximum yield versus optimal purity), but in general it is possible to obtain 10,000 – 40,000 cells per fraction (Figure 3B). Post-sort flow cytometry analysis should reveal >95% purity of each fraction (Figure 3C).
Figure 2: Gating strategy for the identification of the 6 myeloid progenitor fractions. (A) Progressive gating of the live Lin– cells -> singlets (FSC) -> singlets (SSC) -> LKS– cells -> CD34+ FcγRlo ("CMP") and CD34+ FcγRhi ("GMP") fractions -> 6 myeloid progenitor subsets. (B) Summary of surface marker expression by the 6 progenitor fractions. Please click here to view a larger version of this figure.
Figure 3: Representative progenitor yields and purity. (A) Representative flow cytometry plots showing c-Kit and CD11b expression by bone marrow cells before and after lineage depletion, demonstrating enrichment of progenitors (c-Kit+ cells). (B) Cell yields for each progenitor fraction, presented as mean + standard deviation of 30 experiments. Bone marrow cells from up to 20 mice were pooled for each experiment. (C) Representative flow cytometry plots from post-sort analysis, demonstrating the purity of FACS-sorted progenitor fractions. Panel C has been modified from Yáñez et al. 20177. Please click here to view a larger version of this figure.
Laser | Channel | Filter | Mirror |
405 nm | A. | 670/30 | 635LP |
B. | 525/50 | 505LP | |
C. c-Kit-Pacific Blue | 450/50 | ||
488 nm | A. (FSC) | ||
B. Ly6C-PerCP-Cy5.5 | 710/50 | 690LP | |
C. CD34-FITC | 525/50 | 505LP | |
D. SSC | 488/10 | ||
561 nm | A. Sca-1-PE-Cy7 | 780/40 | 750LP |
B. | 710/50 | 690LP | |
C. | 660/20 | 640LP | |
D. | 610/20 | 600LP | |
E. CD115-PE | 582/15 | ||
640 nm | A. FcϒR-APC-Cy7 | 780/60 | 750LP |
B. | 730/45 | 690LP | |
C. Flt3-APC | 670/30 |
Table 1: Antibody panel and flow cytometer configuration.
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 mapping of myelopoiesis pathways, as well as investigation of molecular mechanisms operating at specific stages of myeloid differentiation. The progenitor subsets can be sorted, if desired, for molecular analyses, in vitro and in vivo assessment of differentiation etc.
The protocol was developed using young (6 – 8 weeks old) C57BL/6J mice and is suitable for both male and female mice. We have not characterized the progenitor subsets in older mice. We don't anticipate the markers will change with age, but the relative proportions of the subsets may vary. We have not assessed the progenitor subsets in other mouse strains.
The first application of our modified gating strategy was to provide mechanistic insight into the regulation of myeloid cell fate choice by the transcription factor interferon regulatory factor 8 (IRF8)2. IRF8 was previously reported to be expressed by GMPs (the Weissman "GMP" fraction) and required for monocyte differentiation by promoting monocyte gene expression, suppressing neutrophil gene expression, and promoting neutrophil apoptosis10. Using our modified gating strategy, we demonstrated that IRF8 is not expressed by oligopotent GMPs, but is expressed by both GPs and MPs+cMoPs2. Moreover, we demonstrated that IRF8 regulates GP and MP+cMoP survival and differentiation, rather than production of the lineage-committed progenitors by oligopotent GMPs. Similarly, we recently used our modified gating strategy to re-define the model of myelopoiesis, and demonstrated that GMPs and MDPs produce functionally distinct inflammatory monocytes via 2 independent pathways7.
The lineage depletion step can be performed using a manual magnetic separator instead of an automated magnetic separator, and lineage depletion can also be achieved by flow cytometry using the biotinylated lineage marker antibody cocktail and a fluorophore-conjugated anti-biotin antibody, with gating to select the unstained cells. Lineage depletion excludes most differentiated cells and enriches for c-Kit+ progenitors to minimize the time and costs associated with the subsequent steps (i.e., reduces antibody volumes for flow cytometry, analyzer/sorter time, etc.). Investigators should refer to the manufacturer's datasheet to determine whether the lineage depletion step worked as efficiently as anticipated for the specific kit used. However, it doesn't matter if lineage depletion is not 100% efficient because differentiated (Lin+) cells don't express c-Kit, so any differentiated cells remaining after lineage depletion (as well as other c-Kit– cells) are subsequently excluded by flow cytometry gating. It is also important to note that GPs and MPs express Ly6C but, unlike the Ly6C expressed by monocytes, the progenitor Ly6C (presumably a different isoform) is not detected by the Gr-1 antibody often included in lineage depletion kits, and thus GPs and MPs remain after lineage depletion2.
Good staining is important for all the surface markers, but particularly critical for FcγR to permit accurate gating of the FcγRlo "CMPs" and the FcγRhi "GMPs". Optimal fluorophore choice and antibody dilution are essential for good separation, and an FcγR blocking reagent (commonly used prior to staining for flow cytometry) must not be used. It is also important to note that CD115 is downregulated when the cells are maintained at 4°C or on ice for extended periods, so it is important to work quickly to obtain good staining. We recommend processing the cells within 1 – 2 hours to preserve their integrity. If live cells are not required, stained cells can be fixed and acquired within 48 hours.
If the isolated fractions contain significant numbers of dead cells, it is possible to include a viability dye (e.g., propidium iodide) to more accurately gate live cells, although this would probably necessitate a change in the combination of fluorochrome-conjugated antibodies. If desired, cell counts can be calculated by adding counting beads at a known concentration to the sample for flow cytometry. Counting beads are fluorescent latex beads that can be distinguished from the cells by FSC-A and SSC-A or by their high fluorescence intensity. Cell numbers can be calculated using the following formula:
(number of cell events / number of bead events) x (number of beads added to sample / volume of sample in μL) = concentration of sample as cells/μL
One limitation of the current protocol is that it does not permit separate identification of MPs and cMoPs, which complicates evaluation of these progenitors, investigation of their gene expression and functional properties, etc. For example, an observed increase in MP+cMoP numbers does not indicate whether enhanced monocyte production occurs via the GMP pathway, the MDP pathway, or both. In ongoing studies, we hope to identify surface markers that distinguish between MPs and cMoPs. In the meantime, evaluation of these progenitors by single-cell RNA sequencing7 could be used to profile the relative proportions of MPs and cMoPs.
Single-cell RNA sequencing studies will likely also reveal additional heterogeneity within the 6 progenitor fractions described in this protocol, some of which will reflect different maturation states e.g., GPs that have only recently lost monocyte potential versus GPs that are poised to proceed to the next stage of differentiation. The "GMP" fraction subsets may also contain progenitors with the potential to produce other granulocytes (eosinophils, basophils, and mast cells), but we have not investigated this. As noted in the Introduction, it is also currently unclear whether the CMP-Flt3+ CD115lo, CMP-Flt3– and MDP fractions truly contain the predicted oligopotent progenitors or alternatively comprise a mixture of progenitors with more limited lineage potential, but single-cell RNA sequencing may provide insight into this important question.
A new approach is now required for the identification of subsets of human myeloid progenitors. Weissman and colleagues previously defined myeloid subsets (including "CMPs" and "GMPs") in human bone marrow and cord blood11, but they are similarly heterogeneous. Unfortunately, translation of mouse gating strategies to human progenitors is complicated by notable differences in surface marker expression between human and mouse progenitors; e.g., CD34 expression is limited to myeloid progenitor subsets in the mouse, but more broadly observed on human progenitors including hematopoietic stem cells. Single-cell RNA sequencing studies should, however, also permit the identification of candidate markers to separate the human progenitor subsets.
The authors have nothing to disclose.
This protocol was developed using funds from the Board of Governors Regenerative Medicine Institute at Cedars-Sinai Medical Center (to HSG), a Careers in Immunology fellowship from the American Association of Immunologists (to AY and HSG), and a Scholar Award from the American Society of Hematology (to AY). We thank the Flow Cytometry Core at Cedars-Sinai Medical Center for assistance with FACS sorting.
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 |