This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.
Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin–Sca-1–c-Kit+CD16/32–CD150+CD9dim) and MKp (Lin– Sca-1–c-Kit+CD16/32–CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.
Blood platelets are produced by megakaryocytes. These large polyploid cells are located in the bone marrow and as for all blood cells they are derived from Hematopoietic Stem Cells (HSC)1. The classical pathway of production of megakaryocytes in the bone marrow originates from HSC and involves the generation of different progenitors that progressively restrict their differentiation potential2. The first progenitor signing the commitment to the megakaryocytic lineage is the Megakaryocyte-Erythrocyte Progenitor (MEP), a bipotent progenitor capable of producing both erythroid cells and megakaryocytes3,4,5. The MEP then produces a unipotent progenitor/precursor (MKp) that will differentiate into a mature megakaryocyte capable of producing platelets. The mechanisms involved in the generation of these progenitors, as well as their differentiation and maturation into megakaryocytes are complex and only partially understood. In addition, the heterogeneity of the MEP population in terms of differentiation potential and the intrinsic commitment level of these cells are still unclear. To decipher these processes, it is essential to obtain (or have access to) purified populations of MEP and MKp for fine molecular and single cell analyses.
Several studies have demonstrated particular combinations of cell surface markers for the identification of progenitors committed to the megakaryocytic lineage in the mouse6,7,8. From these a method was devised allowing the purification of MEP and MKp from mice. This method was optimized to obtain cells in adequate number and quality for a large number of assays. With ethical considerations in mind, and in order to minimize the number of animals involved in the experiments, we elicited to harvest the bone marrow from the femur and tibia, and also from the iliac crest. This bone contains a high frequency and number of hematopoietic progenitors and is most of the time damaged during long bone harvesting. Presented here is a detailed method for the reliable collection of this bone.
The second criteria of optimization is to produce highly purified cell populations. Fluorescent Activated Cell Sorting (FACS) is a method of choice in order to obtain purified populations of cells of interest. However, low yields are reached when the cell population of interest is very rare. Enrichment procedures are thus necessary. In this protocol, a negative selection procedure was opted using magnetic beads.
Protocols involving animals were performed in accordance with the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg. Permit Number: E67-482-10).
1. Mouse bone collection
Figure 1: Mouse anatomy. (A) Mouse X-Ray showing the hindlimb bones. Note the space between the pelvic bone and the spine (yellow arrow), where the scissors must be inserted to properly separate the hindlimbs from the body of the mouse (yellow dotted line). (B) Schematic representation of the bone marrow-rich bones of interest. The pelvic bones are depicted in red, the femurs in purple, and the tibias in green. (C) Schematic representation of the mouse pelvic bone. The ilium corresponds to the marrow-rich part of the pelvic bone and is highlighted in red. Please click here to view a larger version of this figure.
2. Magnetic depletion of lineage positive cells
Antibody | Dilution |
Gr-1-biotin | 1:500 |
B220-biotin | 1:500 |
Mac-1-biotin | 1:500 |
CD3-biotin | 1:500 |
CD4-biotin | 1:500 |
CD5-biotin | 1:500 |
CD8-biotin | 1:500 |
TER119-biotin | 1:1000 |
CD127-biotin | 1:500 |
Table 1.
3. Cell sorting of megakaryocyte progenitors by flow cytometry
Tube | Label | Antibody cocktail |
Total Bone Marrow | ||
1 | Unstained control | |
2 | Single stained control | CD45-FITC (1/200) |
3 | Single stained control | CD45-PE (1/200) |
4 | Single stained control | TER119-APC (1/200) |
5 | Single stained control | CD45-PECy7 (1/200) |
6 | Single stained control | CD45-APC-Cy7 biotin (1/200) |
Lin-Pos Fraction | ||
7 | Single stained control | Single stained control. Streptavidin-APC-Cy7 (1/500) |
Lin-Neg Fraction | ||
8 | FMO FITC control | c-kit-APC (1/200) + Sca-1-PE (1/200) + CD16/32-PE (1/200) + CD150-PECy7 (1/200) + Streptavidin-APC-Cy7 (1/500) |
9 | FMO PE control | CD9-FITC (1/200) + c-kit-APC (1/200) + CD150-PECy7 (1/200) + Streptavidin-APC-Cy7 (1/500) |
10 | FMO PECy7 control | CD9-FITC (1/200) + c-kit-APC (1/200) + Sca-1-PE (1/200) + CD16/32-PE (1/200) + Streptavidin-APC-Cy7 (1/500) |
11 | Positive tube for sorting | CD9-FITC (1/200) + c-kit-APC (1/200) + Sca-1-PE (1/200) + CD16/32-PE (1/200) + CD150-PECy7 (1/200) + Streptavidin-APC-Cy7 (1/500) |
Table 2.
Phenotypic analysis of the cells identified as MEP and MKp were performed by flow cytometry. Cells were labeled with fluorescence conjugated antibodies to CD41a and CD42c, classical markers of the megakaryocytic and platelet lineages. Both markers were expressed by the cells of the MKp population while these markers are not yet detected at the surface of the cells of the MEP population (Figure 4Ai,4Aii). Polyploidy is a hallmark of megakaryocytes. The DNA content of the sorted populations was also analyzed and demonstrated that the cells are mostly 2N for the MEP population and a small proportion of the MKp cells are 4N, but higher ploidy are not significantly detected in these populations (Figure 4Aiii).
In order to confirm the identity of the sorted cell populations, several differentiation assays were performed to evaluate their capacity to differentiate toward the megakaryocytic and erythroid lineages. First, semi-solid clonogenic assays were performed to quantify megakaryocytic progenitor (CFU-MK) and erythroid progenitors (BFU-E). CFU-MK were detected in both MEP and MKp populations but not in the other population tested (Figure 4B). BFU-E were not detected in the MKp population but were detected in MEP population and the CD150–CD9dim cell population (Figure 4C).
The differentiation of the sorted cells was also followed in liquid culture in the presence of a low concentration of hematopoietic cytokines. Representative images from microscopic observation on the 3rd day of differentiation show that MEP and MKp produced mainly megakaryocytes that are identified as large cells (Figure 5Aiii,5Aiv). Megakaryocytes were identified using CD41 and CD42c expression and represent 53.9 ± 10.4% and 82.0 ± 2.0% of the cells produced from MEP and MKp cell populations, respectively (Figure 5B). Noticeably, the ploidy of the megakaryocytes produced analyzed using DNA marker Hoescht 33242, was greater for the megakaryocyte derived from MKp population compared to the MEP population suggesting a more mature state (Figure 5C). Finally, the cells produced from each population on the 3rd day were subjected to a proplatelet formation assay9. It was observed that only the cells derived from the MKp population were capable of proplatelet emission in this condition (Figure 5D). This suggests a more advanced maturation stage for the MKp population. Furthermore, when culture duration is extended up to 4-5 days, megakaryocytes generated from MEP will also extend proplatelets.
Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. Please click here to view a larger version of this figure.
Figure 3: Cell sorting gating strategy. (A) Selection of the events corresponding to viable single cells. (B) MEP and MKp population selection. (i) The Lin Neg population is selected from the viable single cell events. (ii) Progenitors expressing c-kit and with low to no expression of Sca-1 or CD16/32 antigen are then selected. (iii) CD9 and CD150 expression levels define four cell populations. MKp are defined as CD9brightCD150+ cells, MEP are defined as CD9dimCD150+. The higher limit for the CD9 expression for the CD9dimCD150+ population is based on the maximum CD9 expression level for the CD150– cells. For the purpose of analysis, CD9dimCD150– cells (Progenitors) and CD9–CD150– (Double Negative: DN) were also sorted. (C) Cell sorting gates are based on Fluorescence Minus One (FMO) controls. (i) FMO control for CD9 gates (ii) FMO control for CD150 gates. Please click here to view a larger version of this figure.
Figure 4: Characterization of the MEP and MKp cell populations. (A) Flow cytometry analysis of (i) CD41 expression, (ii) CD42c expression and (iii) DNA content (Hoechst33342) in the CD9+CD150dim (MEP) and CD9+CD150bright (MKp) cell populations. (B) Quantification of CFU-MK from the sorted cell populations. CD9–CD150– (DN), CD9+CD150– (Prog), CD9+CD150dim (MEP), and CD9+CD150bright (MKp) cell populations were sorted and plated in collagen gel according to the manufacturer's instructions. (C) Quantification of BFU-E from the sorted cell populations. CD9–CD150– (DN), CD9+CD150– (Prog), CD9+CD150dim (MEP), and CD9+CD150bright (MKp) cell populations were sorted and plated in methyl cellulose gel according to the manufacturer's instructions. Please click here to view a larger version of this figure.
Figure 5: Differentiation potential of MEP and MKp. CD9–CD150–(DN), CD9+CD150–(Prog), CD9+CD150dim(MEP), and CD9+CD150bright(MKp) cell populations were cultured for three days in StemSpan medium supplemented with SCF (7.5 ng/mL), Flt-3 (5 ng/mL), IL-6 (1 ng/mL), and TPO (10 ng/mL). (A) Representative images were taken by phase-contrast microscopy. (B) The percentage of CD41+CD42c+ megakaryocytes was then assessed by flow cytometry. N = 3. (C) The ploidy level of the CD41+CD42c+ megakaryocytes was then evaluated with Hoechst by Flow cytometry. N = 3. (D) Cells produced at day 3 from CD9–CD150–(DN), CD9+CD150–(Prog), CD9+CD150dim(MEP), and CD9+CD150bright(MKp) cell populations were harvested and cultured in DMEM medium supplemented with 50 ng/mL TPO, 10 % Fetal Calf Serum, and 100 U/mL hirudin. (i) The proportion of proplatelet-forming megakaryocytes in the culture was determined by microscopic observation. Megakaryocytes were identified based on their size and/or the presence of proplatelets. N = 2. (ii) Representative photograph of a proplatelet bearing megakaryocyte by phase-contrast microscopy. Please click here to view a larger version of this figure.
The method described in this paper allows for the extraction and purification of mouse MEP and MKp. An important parameter in the optimization of the protocol was to obtain sufficient number of cells that would be compatible with most molecular- and cellular-based assays. The general practice of mouse bone collection for hematopoietic cell extraction usually consists in harvesting both the femurs and tibias of each mouse. The pelvic bone, another source of hematopoietic material, is thus often overlooked. The reasons for not collecting the iliac crest is the poor knowledge of the internal anatomy of the mouse skeleton and the fact that users are classically collecting hindlimbs by cutting across or just above the femur head. In addition, it is often assumed that the marrow cells would not be flushed out of the iliac crest bone efficiently due to the presence of trabeculae, which are absent in the central part of the tibia and femur. In this protocol, these two concerns are addressed and a standardized, reliable, and time-effective method is presented that allows for proper flushing of each hindlimb bones, including the pelvic bone. In particular, the use of the iliac bone yields 105 ± 7 x 106 cells per mouse while the classical method usually yields 42 ± 5 x 106 cells. A major benefit of this method is the reduction in the number of animals required to obtain a given number of target cells, thus providing more ethical and cost-effective experimental conditions. This procedure is therefore also applicable for any study requiring bone marrow cell suspension such as isolation of hematopoietic stem cells10 or the analysis of hematopoietic progenitor behavior in semi solid conditions11.
Cell sorting using flow cytometry is a powerful technique with a major advantage in term of purity when compared to magnetic enrichment techniques, but the yield of cell sorting for rare populations can be lower than for more abundant populations. Magnetic depletion of unwanted cells beforehand is therefore a useful method to increase the frequency of the cells of interest. Here, the magnetic depletion procedure differs from the manufacturer's recommendation and takes into consideration the heterogeneity in the expression of the surface markers used to remove the unwanted lineage positive cells. With the typical, one-step protocols, lineage positive cells with the highest expression of surface markers will quickly saturate the magnetic beads. They will prevent the subsequent capture of the remaining labeled cells by competition and steric hindrance, thus significantly reducing the depletion efficacy. To address this problem, a two-step magnetic depletion was designed that allows for the sequential removal of all lineage-positive cells, therefore allowing for stringent depletions suitable for cell sorting.
Another critical parameter to achieve an efficient depletion is the appropriate labeling conditions of the unwanted lineage-positive cells. The antibody titration has therefore been specifically optimized. Using higher concentrations of antibodies will result in excessive rosetting of the magnetic beads and the non-specific depletion of lineage-negative cells of interest. The use of highly purified MEP and MKp cell populations is an important tool in the study of megakaryopoiesis. In order to elucidate the mechanisms controlling this process, the study investigated the role of the cellular microenvironment and have shown that a fetal liver cell stromal cell population would support the differentiation of MKp10. The sorted population could also be used for molecular- or single cell-based analyses. This will be particularly relevant considering the emerging notion of megakaryocyte-biased HSC12,13,14. The production of megakaryocyte directly from the HSC population without the generation of a bipotent progenitor would be an emergency pathway in response to stress13.
The authors have nothing to disclose.
The authors wish to thank Monique Freund, Catherine Ziessel and Ketty for technical assistance. This work was supported by ARMESA (Association de Recherche et Développement en Médecine et Santé Publique), and by Grant ANR-17-CE14-0001-01 to Henri.de la.Salle.
21-gauge needles | BD Microlance | 301155 | |
7AAD | Sigma-Aldrich | A9400 | |
Antibody Gr-1-biotin | eBioscience | 13-5931-85 | Magnetic depletion |
Antibody B220-biotin | eBioscience | 13-0452-85 | Magnetic depletion |
Antibody Mac-1-biotin | eBioscience | 13-0112-85 | Magnetic depletion |
Antibody CD3e-biotin | eBioscience | 13-0031-85 | Magnetic depletion |
Antibody CD4-biotin | eBioscience | 13-9766-82 | Magnetic depletion |
Antibody CD5-biotin | eBioscience | 13-0051-85 | Magnetic depletion |
Antibody CD8a-biotin | eBioscience | 13-0081-85 | Magnetic depletion |
Antibody TER119-biotin | eBioscience | 13-5921-85 | Magnetic depletion |
Antibody CD127-biotin | eBioscience | 13-1271-85 | Magnetic depletion |
Antibody CD45-FITC | eBioscience | 11-0451-85 | Cell sorting |
Antibody CD45-PE | eBioscience | 12-0451-83 | Cell sorting |
Antibody TER119-APC | eBioscience | 17-5921-83 | Cell sorting |
Antibody CD45-PECy7 | eBioscience | 25-0451-82 | Cell sorting |
Antibody CD45-biotin | eBioscience | 13-0451-85 | Cell sorting |
Antibody CD9-FITC | eBioscience | 11-0091-82 | Cell sorting |
Antibody c-kit-APC | eBioscience | 17-1171-83 | Cell sorting |
Antibody Sca-1-PE | eBioscience | 12-5981-83 | Cell sorting |
Antibody CD16/32-PE | eBioscience | 12-0161-83 | Cell sorting |
Antibody CD150-PECy7 | eBioscience | 25-1502-82 | Cell sorting |
Culture medium StemSpan-SFEM | Stemcell technologies | #09650 | |
Dissection pad | Fisher Scientific | 10452395 | |
DPBS | Life Technologies | 14190-094 | |
Ethanol | vWR Chemicals | 83813.360 | |
Forceps | Euronexia | P-120-AS | |
Glass pasteur pipette | Dutscher | 42011 | |
Magnet : DynaMag-5 | Thermo Fisher Scientific | 12303D | |
Magnetic beads: Dynabeads Sheep Anti-Rat IgG | Thermo Fisher Scientific | 11035 | |
Megacult | Stemcell technologies | #04970 | |
MethoCult SF M3436 | Stemcell technologies | #03436 | |
Newborn Calf Serum | Dutscher | 50750-500 | |
Red Cell Lysis solution | BD Bioscience | 555899 | |
Scalpels | Fisher Scientific | 12308009 | |
Scissors | Euronexia | C-165-ASB | |
Sterile 1 mL syringes | BD Bioscience | 303172 | |
Sterile 15mL tubes | Sarstedt | 62.554.502 | |
Sterile 5mL polypropylene tubes | Falcon | 352063 | |
Sterile 5mL polystyrene tubes | Falcon | 352054 | |
Sterile tubes with 70µm cell strainer cap | Falcon | 352235 | |
Sterile petri dish | Falcon | 353003 | |
Streptavidin-APC-Cy7 | BD Biosciences | 554063 | Cell sorting |
Tube roller | Benchmark Scientific | R3005 |