Name: isolation of mouse megakaryocyte progenitors
Uploaded: 2021-05-20T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Megakaryocyte Progenitors at JoVE.com

The authors wish to thank Monique Freund, Catherine Ziessel and Ketty for technical assistance. This work was supported by ARMESA (Association de Recherche et D&#233;veloppement en M&#233;decine et Sant&#233; Publique), and by Grant ANR-17-CE14-0001-01 to Henri.de la.Salle.

Protocols involving animals were performed in accordance with the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Comit&#233; R&#233;gional d&#39;Ethique en Mati&#232;re d&#39;Exp&#233;rimentation Animale Strasbourg. Permit Number: E67-482-10).
1. Mouse bone collection
<ol>
	<li>Sacrifice the animal in compliance with the institutional guidelines. 
	NOTE: The data presented in this manuscript were obtained from C57Bl/6 mice of 8 to 12 weeks old. Th...

     

<ol>
	<li>Kaushansky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+review:+megakaryopoiesis+and+thrombopoiesis.">Historical review: megakaryopoiesis and thrombopoiesis.</a> Blood. 111 (3), 981-986 (2008).</li><li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404 (6774), 193-197 (2000).</li><li>Debili, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+bipotent+erythro-megakaryocytic+progenitor+in+human+bone+marrow.">Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow.</a> Blood. 88 (4), 1284-1296 (1996).</li><li>Forsberg, E. C., Serwold, T., Kogan, S., Weissman, I. L., Passegu&#233;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+evidence+supporting+megakaryocyte-erythrocyte+potential+of+flk2/flt3++multipotent+hematopoietic+progenitors.">New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors.</a> Cell. 126 (2), 415-426 (2006).</li><li>Vannucchi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+bipotent+(erythroid+and+megakaryocytic)+cell+precursor+from+the+spleen+of+phenylhydrazine-treated+mice.">Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.</a> Blood. 95 (8), 2559-2568 (2000).</li><li>Pronk, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+phenotypic,+functional,+and+molecular+topography+of+a+myeloerythroid+progenitor+cell+hierarchy.">Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.</a> Cell Stem Cell. 1 (4), 428-442 (2007).</li><li>Nakorn, T. N., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mouse+clonogenic+megakaryocyte+progenitors.">Characterization of mouse clonogenic megakaryocyte progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (1), 205-210 (2003).</li><li>Ng, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+thrombopoietin+(TPO)-responsive+progenitor+cells+in+adult+mouse+bone+marrow+with+in+vivo+megakaryocyte+and+erythroid+potential.">Characterization of thrombopoietin (TPO)-responsive progenitor cells in adult mouse bone marrow with in vivo megakaryocyte and erythroid potential.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (7), 2364-2369 (2012).</li><li>Strassel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Brouard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+microenvironment+in+the+developing+liver+supports+the+expansion+of+megakaryocyte+progenitors.">A unique microenvironment in the developing liver supports the expansion of megakaryocyte progenitors.</a> Blood Advances. 1 (21), 1854-1866 (2017).</li><li>Boscher, J., Gachet, C., Lanza, F., L&#233;on, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+culture+in+3D+methylcellulose-based+hydrogel+to+improve+cell+maturation+and+study+the+impact+of+stiffness+and+confinement.">Megakaryocyte culture in 3D methylcellulose-based hydrogel to improve cell maturation and study the impact of stiffness and confinement.</a> Journal of Visualized Experiments:JOVE. , (2021).</li><li>Sanjuan-Pla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-biased+stem+cells+reside+at+the+apex+of+the+haematopoietic+stem-cell+hierarchy.">Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.</a> Nature. 502 (7470), 232-236 (2013).</li><li>Haas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-driven+fast-track+differentiation+of+HSCs+into+the+megakaryocytic+lineage.">Inflammation-driven fast-track differentiation of HSCs into the megakaryocytic lineage.</a> Experimental Hematology. 42 (8), 14 (2014).</li><li>Shin, J. 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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...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62498">Isolation of Mouse Megakaryocyte Progenitors</a>. A figure was updated.
Figure 2 was updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62498/62498fig02.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/62498/62498fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62498/62498fig2v2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/62498/62498fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62498">Isolation of Mouse Megakaryocyte Progenitors</a>. A figure was updated.

Erratum: Isolation of Mouse Megakaryocyte Progenitors

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.

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isolation of mouse megakaryocyte progenitors

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.

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 sort...

Watch this Scientific Journal Video about Isolation of Mouse Megakaryocyte Progenitors at JoVE.com

Isolation of Mouse Megakaryocyte Progenitors

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 ...

The isolation of key Megakaryocyte Progenitor populations, allows for the fine analysis of the lineage hierarchy in cell culture on molecular assays, up to the single cell level. This protocol combines a more ethical practice by minimizing the number of animals required, in an efficient process for the cell sorting of MEP and MKP populations. For bone, collection spray the body of an eight to 12 week old C57 black six mouse, with 70%ethanol.Use scissors to make a point five to one centimeter incision of the skin, perpendicular to the spine. And tear the skin around the whole body, by pulling it down. Place the mouse face down on the dissection pad, then locate the pelvic bones by sliding fingers or scissors, along the exposed spine, from top to bottom.To locate the iliac crest, identify the small bump in the lumbar region near the hind limbs. After placing the scissors parallel to the spine, against the vertebrae, and close to the iliac crest bump, proceed to cut the muscles along the side of the spine above the pelvic bone, by sliding the scissors along the vertebrae, down to the tail. Then cut between the vertebrae and the iliac crest, staying as close to the vertebrae as possible.And cut the remaining muscles to detach the limb from the body. Transfer the detached limbs to a clean surface. After discarding the rest of the body in compliance with the institutional guidelines, use forceps and scalpels to expose the pelvic, femoral, and tibial bones, by removing surrounding tissue.Use forceps to hold the distal end of the femur, and carefully dislocate the femoral head from the pelvic bone, by gently slicing the muscles around the articulation with a scalpel. Scrape off the remaining muscle from the pelvic bone, and cut in the middle of the cavity that held the femoral head. Keep the ilium and discard the triangular thin side of the bone.Using a scalpel, remove the residual tissues around the ilium, and place the cleaned bone in sterile PBS supplemented with 2%Newborn Calf Serum. Then use scissors to cut off the foot from the leg at the ankle. Holding the lower part of the tibia with the forceps, scrape the muscle up toward the knee.Discard the fibula. Then make a cut across the tibial plateau with the scalpel, and place the tibia in sterile PBS 2%NBCS. After removing the residual tissues around the femur, hold the upper side of the femur with forceps, and place the scalpel blade at the base of the kneecap.Apply a force toward the kneecap, parallel to the femur to detach the kneecap. Then place the femur, in sterile PBS 2%NBCS. In a laminar flow cabinet, transfer the bones to a sterile Petri dish filled with sterile PBS 2%NBCS.Use a scalpel to cut off the head of the femurs. Fill a one milliliter syringe with sterile PBS 2%NBCS, and attach a 21 gauge needle, to the outlet. Then fill a five milliliter polypropylene tube with two milliliters of sterile PBS 2%NBCS.Holding the femur with forceps, insert the needle in the groove left, after the kneecap removal by applying rotation, ensuring that the needle is completely inserted into the bone, up to the bevel. After the needle insertion, transfer the bone with the needle into the tube containing two milliliters of PBS 2%NBCS. Then dispense and aspirate the PBS 2%NBCS, from the syringe until the bone is clear.Remove the needle from the femur, and insert it into the hole at the opposite side, where the femur head was. After dispensing and aspirating the buffer, discard the bone. For the iliac crest and tibia, use forceps to hold the bone and gently insert the needle in the open side, by applying rotation.Ensuring that the needle is completely inserted into the bone, up to the bevel. Then transfer the bone with the needle into the tube containing two milliliters of PBS 2%NBCS. Dispense and aspirate the PBS 2%NBCS from the syringe until the bone is clear.Pass all the the cell suspension through a 40 micron cell strainer cap, placed onto a sterile five milliliter polystyrene tube, and add 500 microliters of PBS 2%NBCS. Set aside 100 microliters of the cell suspension as total bone marrow. Then store it on ice for the staining procedure.Pellet the filtered suspension by centrifugation. After discarding the supernatant, resuspend the pellet in a freshly prepared primary antibody cocktail, with the incubation on ice, for 30 to 45 minutes. Set aside 10 microliters of the cell suspension into a sterile five milliliter polystyrene tube, labeled Lin Pores Fraction, and add 90 microliters of PBS 2%NBCS.Meanwhile, prepare a beads for magnetic depletion, by resuspending them in the vial, with thorough vortexing for 30 seconds. Transfer a volume of beads corresponding to two beads per target cell, into a five milliliter polypropylene tube, and wash the beads twice, with PBS 2%NBCS, by placing tube on the magnet. After removing the washing buffer with a sterile glass pasteur pipette, resuspend the beads in 500 microliters of sterile PBS 2%NBCS.For the first step of magnetic depletion, add 250 microliters of beads onto the pellet of washed cells, and incubate with gentle mixing for five minutes on ice. Then add two milliliters of sterile PBS 2%NBCS, with gentle mixing, and place the tube in the magnet for two minutes. Proceed to collect the non-magnetic fraction with a sterile glass pasteur pipette.And for the second step of magnetic depletion, add it to the remaining 250 microliters of magnetic beads. Place the tube sealed with paraffin on a tube roller, for 20 minutes at four degrees Celsius. Then add two milliliters of sterile PBS 2%NBCS with gentle mixing.And place the tube with the magnet for two minutes. Collect the non-magnetic fraction into a sterile five milliliter polypropylene tube, labeled, non-magnetic Lin Neg Fraction, with a sterile glass pastreur pipette, and proceed with cell sorting megakaryocytes progenitors, as described in the text manuscript. For the gating strategy for cell sorting, in the Lin Neg population, the cells expressing C-kit, and a low to no expression of Sca-1 or CD16/32 antigen are selected.In this population, MEP are identified as CD150 positive and CD9 dim cells. The MKP as CD150 positive, and CD9 bright cells. In the representative Flow Cytometry Analysis, cells identified as MEP and Mkp, were labeled with fluorescence conjugated antibodies for CD41a, and CD42c classical markers, of the megakaryocytic and platelet lineages.Both markers were expressed by the cells of the MKp population, and were not detected at the surface of the cells of the MEP population. The DNA content of the sorted megakaryocytes, demonstrated that the cells are mostly 2n for the MEP population. And a small portion of the MKp cells are foreign.Higher ploidy cells were not significantly detected in these populations. In semi-solid clonogenic assays, CFU-MK was detected in both MEP and MKp populations. BFU-E was not detected in the MKp population, but detected in MEP and, the CD150 negative CD9 dim progenitor cell population.Microscopic observation on the third day of differentiation, shows that MEP and MKp produced mainly megakaryocytes, identified as large cells. Megakaryocytes obtained after three days of culture, were identified using CD41 and CD42c expression, and represent 54, and 82%of the cells produced from MEP and MKp cell populations, respectively. The ploidy of the megakaryocytes produced, was greater from the megakaryocyte derived from the MKp population, compared it to the MEP population.Only the cells derived from the MKp population, were capable of proplatelet emission, suggesting a more advanced maturation stage for the MKp population. The use of the pelvic bone, drastically increases to cell number, while two-step magnetic depletion improves the efficacy of the lineage depletion. This protocol allows for the subsequent culture of MEP and MKp population of single cells, which, together with transcriptomic and proteomic analysis, will certainly have to decipher the mechanisms involved, implemented biogenesis.

Research

JoVE Journal

Biology

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Mouse Dorsal Forebrain Explant Isolation

Isolation of Genomic DNA from Mouse Tails

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...