A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.
The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation.
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric ‘forward-scatter’ parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.
1. Harvesting of tissues
2. Preparation of spleen cells
3. Preparation of bone-marrow cells
4. Preparation of fetal liver cells
5. Antibody staining for flow cytometry
6. Flow-cytometric sorting
7. Representative Results:
CD71/Ter119 staining of adult bone-marrow or spleen identifies a developmental sequence of four subsets, labeled ProE, EryA, EryB and EryC (Figure 1) 1. Morphologically, these correspond to increasingly mature erythroblasts. Figure 1 illustrates the gating sequence at the data analysis stage, which discards very small event (including nuclei, red cells), aggregated cells and dead cells.
Expression of cell-surface proteins may be measured simultaneously for each of these subsets, by adding the relevant antibodies at the same time as Ter119 and CD71 staining. Figure 1 shows an example of cell-surface expression of the death receptor Fas 1. This measurement was carried out in mice injected with Epo, or in control mice injected with saline. It is apparent that Epo suppresses Fas expression in the EryA population in vivo 1.
Expression of intracellular proteins or cell cycle status may also be measured for cells in each subset. Figure 2 illustrates representative cell cycle analysis of freshly harvested bone marrow cells. These measurements require, in addition to cell surface staining with CD71 and Ter119, the fixation and permeabilization of cells for intracellular labeling (see Discussion section).
In fetal liver, non-erythroid cells are first excluded by gating on ‘Lin-‘ cells that are negative for CD41, Mac-1, Gr-1, B220 and CD3 (Figure 3). The remaining cells are sub-divided into 6 subsets, S0 to S5. The precise pattern of cells in fetal liver is dependent on embryonic age (see Discussion section). A representative cell cycle analysis of the S3 subset in E13.5 fetal liver is shown (Figure 4).
Figure 1. The CD71/Ter119 erythroid subsets in mouse spleen. A. Gating strategy: Spleen cells were processed and labeled with antibodies directed at CD71, Ter119 and Fas. This figure shows the analysis strategy following the data acquisition step. Histogram I shows all acquired events. The diagonal gate represents events that are likely to be single cells, excluding doublets or larger aggregates. Cells in this gate are further analyzed in histogram II. Here very small events, likely nuclei or debris, are excluded. The gated cells are shown in histogram III, where DAPI-positive cells, that are likely membrane-permeable apoptotic cells, are excluded from further analysis. Histogram IV shows the resulting population of viable spleen cells. The ProE gate contains CD71highTer119intermediate cells. Ter119high cells are further analyzed in histogram V. Here CD71high cells are subdivided into less mature, large ‘EryA’ erythroblasts (CD71highTer119highFSChigh) and smaller, more mature ‘EryB’ erythroblasts (CD71highTer119highFSClow). The most mature erythroblast subset is EryC (CD71lowTer119highFSClow). Histogram VI shows cell-surface Fas expression, specifically in the EryA subset, in mice in the basal state (injected with saline), and mice injected with a single dose of Epo. Staining with Fas antibody was carried out simultaneously with the CD71/Ter119 staining. B. Cytospin preparations of cells sorted from each of the indicated subsets. Cells were stained with Giemsa and with Diaminobenzidine, the latter generates a brown stain with hemoglobin. Cytospin data was originally published in Liu et al., Blood. 2006 Jul 1;108(1):123-33. Epub 2006 Mar 9.
Figure 2. Cell cycle analysis of CD71highTer119high erythroblasts in mouse bone marrow. Mice were injected intraperitoneally with BrdU, and spleen or bone-marrow were harvested 30 to 60 minutes later. Cells were fixed and permeabilized and in addition to being stained for CD71 and Ter119, were stained for BrdU incorporation into their replicating DNA with a monoclonal antibody directed at BrdU (fixation, permeabilization and BrdU-staining protocol was according to manufacturer’s instruction). BrdU-positive cells are in S-phase of the cycle. Interphase cells are BrdU-negative and may be resolved into G1 or G2/M phases, using the DNA dye 7AAD.
Figure 3. CD71/Ter119 erythroid subsets in mouse fetal liver. A. Gating strategy: Fetal liver cells were labeled for CD71, Ter119, and a cocktail of FITC –labeled antibodies directed at non-erythroid lineage markers (‘Lin’). Viable cells (7AAD-negative) were analyzed for Lin expression, and the Lin- cells are further subdivided into the S0 to S5 erythroid subsets. Younger, E13 fetal liver is composed of less mature erythroblasts, shown by the absence of cells in the mature S4/S5 subsets. B. Cytospin preparations of cells sorted from each of the indicated subsets. Cells were stained with Giemsa and with Diaminobenzidine, the latter generates a brown stain with hemoglobin. Cytospin data was originally published in Pop et al., PLoS Biol 8(9): e1000484. doi:10.1371/journal.pbio.1000484 .
Figure 4. Cell cycle analysis of fetal liver erythroid subsets. Pregnant mice were injected with BrdU, and fetal livers were harvested 30 to 60 minutes later, fixed, permeabilized, and stained with antibodies against CD71, Ter119 and BrdU. Cell cycle status of S3 cells is shown.
The flow-cytometric methodology allows simultaneous investigation of any cellular function that may be detected with a fluorescence-conjugated specific antibody or ligand, including cell surface markers, protein expression, cell survival, cell signaling using phospho-specific antibodies 3 and cell cycle status. These measurements may be made in each of a number of differentiation-stage specific subsets, in the context of freshly isolated erythropoietic tissue. This method therefore allows assessment of functional and molecular changes at different levels of the erythropoietic system, in response to a wide range of erythropoietic stimuli or as a result of genetic mutations.
No antibody is without cross reactivity, and cross reactivity may be tissue- specific. It is therefore important to verify, even for previously tested antibodies, their specificity in the context of erythropoietic tissue, using either a null or knock-down cell model.
Cells from specific erythroid subsets may be sorted for RNA or transcriptome analysis. Sort experiments should use low sorting pressures and wide nozzles, in order to minimize the shear stress on the cells. We recommend checking cell purity and viability following each sorting experiment. Of note, in the case of multiparameter flow-cytometry, the true background for each color is its ‘FMO’ control (see 5.3), which includes, in addition to autofluorescence, the background due to spectral overlap from all other colors. At the analysis stage, a subset-specific ‘FMO’ control needs to be used.
Differentiation stage of erythroblasts within the flow-cytometrically defined subsets
The flow-cytometric ProE/EryA/EryB/EryC subsets are defined in terms of cell-surface marker expression and forward scatter. While it is likely that each of these subsets corresponds to approximately the same morphological erythroblast differentiation stage in a wide variety of mouse models, we recommend verifying this when examining a new mouse model. Cells from each of the flow-cytometrically-defined subsets should be sorted and cytospin preparations examined for morphological staging of erythroblasts.
Although the CD71/Ter119 subsets each contain erythroblasts of similar differentiation stage, there remains a degree of heterogeneity within each subset. In the first application of the CD71/Ter119 method, we divided Ter119+ cells into regions I to IV based on their CD71 expression. The precise borders between these regions were determined arbitrarily 4. We subsequently added cell size information to the analysis, in the form of the forward scatter parameter. This allowed us to divide Ter119high cells into subsets by following natural population contours 1 (Figure 1). This approach resulted in populations of more uniform maturation and in more reproducible results, and has been recently adapted by other investigators5,6,7. The EryA subset may be sub-divided further where desired7. One group suggested the use of CD44 in place of CD718. Although CD44 is less useful in resolving early erythroblast stages, it may resolve later erythroblast subsets with more precision. Both markers may therefore be used, or alternatively, their choice may depend on the specific subsets of interest.
An alternative strategy employs multispectral imaging using the ImageStream technology (Amnis Corporation, Seattle, WA)9. It allows the simultaneous and rapid acquisition of both morphological and flow-cytometric data on many thousands of cells. It is likely to become the ‘gold standard’ with respect to molecular analysis of stage-specific erythroblasts, since the morphological criteria by which differentiation stage is defined may be measured directly. However, at the present time this technology is less widely available than conventional flow cytometry, and suffers from two drawbacks: it does not allow cell sorting; and it is limited to a smaller number of flow-cytometric parameters.
Intracellular antigens
Detection of intracellular proteins or BrdU requires cell fixation and permeabilization. The precise fixation and permeabilization procedure depends on the intracellular antigen in question. We use the LIVE/DEAD Fixable Dead Cell Stain (Molecular Probes) during the fixation procedure, to distinguish viable from dead cells. Of note, permeabilization with detergents usually impacts the Ter119 signal, which is partially detergent soluble. We overcome this difficulty by using gentle detergents (such as the saponin-based ‘perm/wash’ buffer from BD Biosciences). We also stain for Ter119 both prior to, and following, the fixation & permeabilization procedure, in order to optimize the Ter119 signal. Alternatively, it is possible to sort viable cells from each of the CD71/ Ter119 subsets first, and carry out fixation and permeabilization separately on purified cells from each subset (e.g. see the cell cycle analysis in fetal liver) 10.
Fetal liver CD71/Ter119 subsets
The CD71/Ter119 staining pattern in fetal liver is dependent on embryonic age 2 (Figure 3). We subdivide fetal liver cells into 6 subsets, S0 to S5 10. At the onset of definitive erythropoiesis in fetal liver on embryonic day 11 (E11), cells are concentrated in subsets S0 and S1 and are largely erythroid colony-forming cells (CFU-e). With embryonic development, CFU-e cells differentiate into proerythroblasts and maturing erythroblasts and gradually populate subsets S2 to S5 (Figure 3).
Subsets S1 to S5 are composed almost entirely of erythroid cells of the definitive lineage. These subsets are absent in the EpoR-/- fetal liver. A small number of Ter119+ cells in fetal liver correspond to the primitive (yolk sac) erythroid lineage. These cells are apparent in EpoR-/- fetal liver, where no definitive lineage erythroblasts arise, but by E13.5 form less than 1% of Ter119+ cells in wild-type fetal liver 10.
The S0 subset is heterogeneous. At E13.5, 70% of S0 cells are erythroid cells at the CFU-e stage, just prior to the onset of EpoR dependence 10. The remainder are earlier progenitors as well as cells of other hematopoietic lineages, principally megakaryocytes and macrophages; these cells may be sorted or gated out 10 (Figure 3).
Interpretation of changes in the frequency of erythroid subsets
Changes in the frequency of erythroid subsets should be interpreted with care. A change in frequency of cells in any given subset may be due to their altered apoptotic rate, altered transit time through that subset, or alternatively may be due to changes in the number of cells in other subsets. A common cause for increased frequency of early erythroblast subsets ProE and EryA is erythropoietic stress of multiple etiologies1. Similar findings have been noted in the 1960’s by inspecting erythroblast morphologies during the stress response 11. The precise reason for the increase in the relative frequency of earlier precursors during stress is not clear, but in part may be due to the improved survival of these precursors during stress 1.
The authors have nothing to disclose.
We thank the UMass flow cytometry core: Richard Konz, Ted Giehl, Barbara Gosselin, Yuehua Gu and Tammy Krupoch. This work was funded by NIH/NHLBI RO1 HL084168 (M.S.) and NIH CA T32-130807 (J.R.S.). Core resources supported by the Diabetes Endocrinology Research Center grant DK32520 were also used.
Name of the reagent | Company | Catalogue number |
---|---|---|
Fas-biotin | BD Pharmingen | 554256 |
Streptavidin-APC | Molecular Probes | S868 |
40 μm sterile cell strainer | Fisherbrand | 22363547 |
Polystyrene round-bottom tubes for FACS staining | BD Falcon | 352008 |
U-bottom 96 well plate | BD Falcon | 353910 |
ChromePure Rabbit IgG | Jackson ImmunoResearch | 015-000-003 |
CD71-FITC (stock 0.5mg/ml) | BD-Biosciences | 553266 |
Ter119-PE (stock 0.2mg/ml) | BD-Biosciences | 553673 |
7AAD | BD-Biosciences | 559925 |
DAPI powder | Roche | 236276 |
FITC Rat Anti-Mouse CD41 MWReg30 | BD Pharmingen | 553848 |
FITC Rat Anti-Mouse CD45R/B220 RA3-6B2 | BD Pharmingen | 553087 |
FITC Rat Anti-Mouse CD411b/Mac-1 M1/70 | BD Pharmingen | 557396 |
FITC Rat Anti-Mouse Ly-6G and Ly-6C (Gr-1) RB6-8C5 | BD Pharmingen | 553126 |
FITC Hamster Anti-Mouse CD3e 145-2C11 | BD Pharmingen | 553061 |
APC BrdU Flow kit | BD Pharmingen | 557892 |
Annexin V-biotin | BD Pharmingen | 556418 |