Name: isolation of mouse megakaryocyte progenitors
Uploaded: 2021-05-20T13:00:00.000+00:00
Duration: 10 min 30 s
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. The number of cells obtained, and the frequency of cited populations may vary with age and mouse strain.</li>
	<li>Spray the body with 70% ethanol.</li>
	<li>Using scissors, make a 0.5-1 cm incision of the skin perpendicular to the spine and tear the skin around the whole body. Pull down the skin from the lower body and remove the skin.</li>
	<li>Place the animal on the dissection pad, face down. Locate the pelvic bones by sliding your fingers along the exposed spine from the top to the bottom. To locate the iliac crest, identify the small bump in the lumbar region near the hindlimbs (the anterosuperior region of the pelvic bone). Figure 1A,B presents a schematic representation of the mouse anatomy.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62498/62498fig01.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62498/62498fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Place 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 all the way down to the tail. 
	NOTE: This first section of muscles can also be performed using a scalpel blade.</li>
	<li>Place the scissors parallel to the spine and proceed to cut between the vertebrae and the iliac crest, as indicated by the yellow dotted line on Figure 1A. Make sure to remain as close to the vertebrae as possible. Cut the remaining muscles to detach the limb from the body. 
	NOTE: There should be little to no resistance.</li>
	<li>Repeat on the other side to detach the second limb.</li>
	<li>Transfer the limbs on a clean surface and discard the rest of the body in compliance with the institutional guidelines.</li>
	<li>Expose the pelvic, femoral, and tibial bones by removing as much surrounding tissue as possible with the forceps and the scalpels.</li>
	<li>Proceed to carefully dislocate the femoral head from the pelvic bone by holding the distal end of the femur with the forceps while gently slicing the muscles around the articulation with the scalpels. Wiggle the bones to facilitate the dislocation.</li>
	<li>Scrape off the remaining muscle from the pelvic bone and cut with a scalpel in the middle of the cavity that did hold the femur head. The ilium is kept as it is rich in hematopoietic progenitors while the triangular very thin side of the bone is discarded, as shown in Figure 1C.</li>
	<li>Remove the residual tissues around the ilium with the scalpel and place the cleaned bone in sterile PBS supplemented with 2% Newborn Calf Serum (PBS-2%NBCS).</li>
	<li>Using scissors, cut off the foot from the leg at the ankle.</li>
	<li>Hold the lower part of the tibia with the forceps and scrape the muscle up toward the knee. Discard the fibulae and cut across the tibial plateau with the scalpel. Place the tibia in sterile PBS-2%NBCS.</li>
	<li>Remove the residual tissues around the femur with scalpels.</li>
	<li>Hold the upper side of the femur with forceps; place the scalpel blade at the base of the kneecap. Apply a force toward the kneecap parallel to the femur until the detachment of the kneecap. Place the femur in sterile PBS-2%NBCS. Removal of the kneecap provides a clean access for inserting the needle for marrow flushing.</li>
</ol>
2. Magnetic depletion of lineage positive cells
<ol>
	<li>In a laminar flow cabinet, transfer the bones in a sterile Petri dish filled with sterile PBS-2%NBCS.</li>
	<li>With a scalpel cut off the head of the femurs.</li>
	<li>Fill a 1 mL syringe with sterile PBS-2%NBCS and attach a 21 G needle to the outlet.</li>
	<li>Fill a 5 mL polypropylene tube with 2 mL of sterile PBS-2%NBCS.</li>
	<li>Hold the femur with the forceps; gently insert the needle in the groove left after the kneecap removal. Apply rotation to the needle while inserting to avoid plugging of the needle. Ensure that the needle is completely inserted into the bone up to the bevel.</li>
	<li>Transfer the bone with the needle into the tube containing 2 mL of PBS-2%NBCS. Dispense and aspirate the PBS-2%NBCS from the syringe until the bone is clear.</li>
	<li>Remove the needle from the femur and insert it in the hole at opposite side where the femur head was. Dispense and aspirate the buffer again and discard the bone.</li>
	<li>For the iliac crest and tibia, hold the bone with the forceps; gently insert the needle in the open side. Apply rotation to the needle while inserting to avoid plugging of the needle. Ensure that the needle is completely inserted into the bone up to the bevel. Transfer the bone with the needle into the tube containing 2 mL of PBS-2%NBCS. Dispense and aspirate the PBS-2%NBCS from the syringe until the bone is clear. Discard the bones. 
	NOTE: Bones from up to three mice can be flushed into the same tube. Pool the cell suspensions.</li>
	<li>Pass the pooled cell suspension through a 40 &#181;m cell strainer cap placed onto a sterile 5 mL polystyrene tube.</li>
	<li>Proceed to count the cells. 
	NOTE: Cell count can be performed with any hemocytometer, using Trypan Blue for viability assessment, or with any automated cell counter. One mouse typically yields 105 &#177; 7 x 106 cells.</li>
	<li>Take aside 100 &#181;L of the cell suspension as Total Bone Marrow, add 500 &#181;L of PBS-2%NBCS and save it on ice for the staining procedure.</li>
	<li>Pellet the filtered suspension by centrifugation at 400 x g for 5 min at 4 &#176;C and discard the supernatant. 
	NOTE: Red blood cells can be lysed by resuspending the pellet in freshly prepared Lysis Solution (1/10th in dH2O). Incubate for 5 min until the suspension becomes clear and bright red and add 10 volumes of sterile PBS. Proceed to wash the cells in PBS-2%NBCS by centrifugation at 400 x g for 5 mins at 4 &#176;C. Be careful when removing the supernatant as the cell pellet is very loose. Perform a second wash with PBS-2%NBCS by centrifugation at 400 x g for 5 min at 4 &#176;C and proceed to step 2.13.</li>
	<li>Resuspend the cell pellet in freshly prepared primary antibody cocktail with a ratio of 100 &#181;L per 1 x 107 cells. Incubate on ice for 30-45 min.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Gr-1-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>B220-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mac-1-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>CD3-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>CD4-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>CD5-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>CD8-biotin</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>TER119-biotin</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>CD127-biotin</td>
			<td>1:500</td>
		</tr>
	</tbody>
</table>
Table 1.
<ol start="14">
	<li>Take aside 10 &#181;L of the cell suspension into a sterile 5 mL polystyrene tube labeled Lin-Pos Fraction. Add 90 &#181;L of PBS-2%NBCS and save it on ice for the staining procedure.</li>
	<li>Proceed to wash the cells twice with sterile PBS-2%NBCS by centrifugation at 400 x g for 5 min at 4 &#176;C. Make sure to do the last wash in a sterile 5 mL polypropylene tube.</li>
	<li>During the washing steps, prepare the beads for the magnetic depletion.
	<ol>
		<li>Resuspend the beads in the vial by thoroughly vortexing for 30 s.</li>
		<li>Transfer a volume of beads corresponding to two beads per target cell into a 5 mL polypropylene tube.</li>
		<li>Wash the beads twice with PBS-2%NBCS by placing the tube on the magnet and removing the washing buffer using a sterile glass Pasteur pipette.</li>
		<li>Resuspend the beads in 500 &#181;L of sterile PBS-2NBCS%.</li>
	</ol>
	</li>
	<li>Resuspend the pellet of labeled cells in 250 &#181;L of beads and mix gently for 5 min on ice. Add 2 mL of sterile PBS-2%NBCS and mix gently. Do not shake the tube.</li>
	<li>Place the tube onto the magnet for 2 min.</li>
	<li>Proceed to collect the non-magnetic fraction with a sterile glass Pasteur pipette and add it onto the remaining 250 &#181;L of magnetic beads. Seal the tube with parafilm.</li>
	<li>Place the tube on a tube roller for 20 min at 4 &#176;C.</li>
	<li>Add 2 mL of sterile PBS-2%NBCS and mix gently. Do not shake the tube.</li>
	<li>Place the tube in the magnet for 2 min.</li>
	<li>Proceed to collect the non-magnetic fraction into a sterile 5 mL polypropylene tube labeled Lin-Neg Fraction with a sterile glass Pasteur pipette.</li>
	<li>Pellet the cells by centrifugation at 400 x g for 5 min at 4 &#176;C and remove the supernatant.</li>
	<li>Resuspend the non-magnetic cells in 500 &#181;L of sterile PBS-2%NBCS.</li>
	<li>Proceed to count the cells. 
	&#8203;NOTE: One mouse typically yields 3.9 &#177; 1.1 x 106 cells. Typical lineage staining pre- and post- depletion are presented in Figure 2B.</li>
</ol>
3. Cell sorting of megakaryocyte progenitors by flow cytometry
<ol>
	<li>Take the tubes labeled Total Bone Marrow, Lin-Pos Fraction, and Lin-Neg Fraction.</li>
	<li>Proceed to split the content of the tube Total Bone Marrow equally into six sterile 5 mL polystyrene tubes. Label the tubes with the numbers 1-6.</li>
	<li>Proceed to label the tube Lin-Pos Fraction with the number 7.</li>
	<li>Proceed to split the content of the tube Lin-Neg Fraction as follows.
	<ol>
		<li>Transfer 50 &#181;L into a sterile 5 mL polystyrene tube containing 250 &#181;L of sterile PBS-2%NBCS. Then, split its content equally into 3 sterile 5 mL polystyrene tubes. Label these tubes with the numbers 8-10.</li>
		<li>The remaining 450 &#181;L of Lin-Neg Fraction cell suspension corresponds to the tube with the number 11.</li>
	</ol>
	</li>
	<li>Add the antibodies to the tubes as described in Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tube</td>
			<td>Label</td>
			<td>Antibody cocktail</td>
		</tr>
		<tr>
			<td colspan="3">Total Bone Marrow</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Unstained control</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>Single stained control</td>
			<td>CD45-FITC (1/200)</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Single stained control</td>
			<td>CD45-PE (1/200)</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Single stained control</td>
			<td>TER119-APC (1/200)</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Single stained control</td>
			<td>CD45-PECy7 (1/200)</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Single stained control</td>
			<td>CD45-APC-Cy7 biotin (1/200)</td>
		</tr>
		<tr>
			<td colspan="3">Lin-Pos Fraction</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Single stained control</td>
			<td>Single stained control. Streptavidin-APC-Cy7 (1/500)</td>
		</tr>
		<tr>
			<td colspan="3">Lin-Neg Fraction</td>
		</tr>
		<tr>
			<td>8</td>
			<td>FMO FITC control</td>
			<td>&#160;c-kit-APC (1/200) + Sca-1-PE (1/200) + 
			CD16/32-PE (1/200) + CD150-PECy7 (1/200) + Streptavidin-APC-Cy7 (1/500)</td>
		</tr>
		<tr>
			<td>9</td>
			<td>FMO PE control</td>
			<td>CD9-FITC (1/200) + c-kit-APC (1/200) + CD150-PECy7 (1/200) + Streptavidin-APC-Cy7 (1/500)</td>
		</tr>
		<tr>
			<td>10</td>
			<td>FMO PECy7 control</td>
			<td>CD9-FITC (1/200) + c-kit-APC (1/200) + Sca-1-PE (1/200) + 
			CD16/32-PE (1/200) + Streptavidin-APC-Cy7 (1/500)</td>
		</tr>
		<tr>
			<td>11</td>
			<td>Positive tube for sorting</td>
			<td>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)</td>
		</tr>
	</tbody>
</table>
Table 2.
<ol start="6">
	<li>Incubate on ice for 30-45 min in the dark.</li>
	<li>Wash the cells with sterile PBS-2%NBCS by centrifugation at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cell pellets as follows.
	<ol>
		<li>For the tubes 1 to 10, resuspend the pellet in 300 &#181;L of sterile PBS-2%NBCS supplemented with 7AAD (2.5 &#181;g/mL final) (PBS-7AAD). 
		CAUTION: 7AAD is a DNA intercalant and must therefore be handled with appropriate PPE (gloves).</li>
		<li>For tube 11, resuspend the pellet in sterile PBS-7AAD at a maximum concentration of 5 x 106 cells per mL and a minimum volume of 1 mL.</li>
	</ol>
	</li>
	<li>Prepare two polypropylene collection tubes labeled MEP and MKp containing 2 mL of PBS-2%NBCS. 
	NOTE: Alternatively, cells can be collected into culture medium or cell lysis buffer depending on the subsequent application for the sorted cells. The use of polystyrene tubes is not recommended because of possible interference with the charged droplets containing the cells of interest.</li>
	<li>Keep all tubes on ice in the dark.</li>
	<li>Proceed to the cell sorter set-up.
	<ol>
		<li>Use the tubes 1-7 to set-up voltage and compensation, tubes 7-10 to determine the sorting gates for the cell populations of interest and tube 11 for cell sorting.</li>
	</ol>
	</li>
	<li>The first steps of the gating strategy aim to exclude doublets and dead cells from the analysis, as described in Figure 3. Identify single viable cells and display SSC-vs Lin-APC-Cy7 dot plot to confirm the efficiency of the lineage depletion. From the Lin- cells a gate is set to select cells positive for c-kit&#160;and negative or dim for Sca-1 and CD16/32. A CD9 vs CD150 expression dot plot for the selected cells allows the identification of four populations. 
	NOTE: MEP and MKp cells are both positive for CD150. Three levels of expression for CD9 can be defined (neg, dim, and high). MKp express high level of CD9 and MEP express CD9 at an intermediate fluorescence intensity level. MEP population corresponds to Lin- c-Kit+ Sca-1-CD16/32-/dim CD150+ CD9dim and MKp population corresponds to Lin- c-Kit+ Sca-1-CD16/32-/dim CD150+ CD9bright. The discrimination between CD9 high and CD9 dim populations for the CD150 positive cells is set based on the maximum level of CD9 expression in the CD150 negative population. One mouse typically yields 5.3 &#177; 0.6 x 103 MKp and 27.2 &#177; 2.4 x 103 MEP.</li>
</ol>

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The authors declare no competing financial interests.

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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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...

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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 ...

isolation-of-mouse-megakaryocyte-progenitors

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

6.3K Views.  Université de Strasbourg, INSERM UMR S1255, EFS Grand-EST. This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Video: Isolation of Mouse Megakaryocyte Progenitors

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Immunology and Infection

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

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 &#34;contaminating&#34; 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.

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 ...

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 constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

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

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Neuroscience

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Medicine

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

It is now acknowledged that the three-dimensional environment of cells can play an important role in their behavior, maturation and/or differentiation. This protocol describes a three-dimensional cell culture model designed to study the impact of physical containment and mechanical constraints on megakaryocytes.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D ...