Name: Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants (Video) | JoVE Journal
Uploaded: 2021-05-20T15:00:00.000+00:00
Duration: 5 min 58 s
Description: Learn a rapid explant method to visualize proplatelet formation from mouse megakaryocytes in native bone marrow. This protocol enables real-time observation and quantitative analysis of megakaryocyte behavior for hematology research.

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5 minutes

The authors wish to thank Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert for technical assistance. This work has been supported by ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 and ANR-18-CE14-0037.

All animal experiments were performed in accordance with European standards 2010/63/EU and 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).
1. Preparation of reagents
<ol>
	<li>Prepare reagents as described in Table 1.
	<ol>
		<li>For the Stock I, dissolve each powder separately. Ensure that the osmolarity of the preparation is higher than 295 mOsm/L. This solution can be stored at 4 &#176;C for one year.</li>
		<li>For the Tyrode&#39;s buffer preparation, make the solution as described in Table 1, adjust the volume to 100 mL with distilled water and add 0.1 g anhydrous D (+) sucrose. Adjust to pH 7.3 using 1 N HCl as needed and the osmolarity to 295 mOsm/L. To prevent bacterial growth, add penicillin G at 10 U/mL final concentration and streptomycin sulfate at 0.29 mg/mL final concentration. Filter the final solution through 0.22 &#181;m pores.</li>
	</ol>
	</li>
</ol>
2. Preparation of the experimental set up
<ol>
	<li>On the day of the experiment, warm the Tyrode&#39;s buffer at 37 &#176;C, and turn on the heating chamber of the microscope to bring the temperature to 37 &#176;C.</li>
	<li>Prepare all the necessary tools, such as a timer, incubation chambers, 5 mL syringes, 21 G, forceps, razor blade, Pasteur pipettes, glass slides, and 15 mL centrifuge tube (Figure 1A).</li>
</ol>
3. Isolation of the mouse bone marrow
<ol>
	<li>Euthanize C57BL/6 mice aged 8-12 weeks by CO2 asphyxiation and cervical dislocation. This should be done quickly by a competent and qualified person.</li>
	<li>To avoid microbial contamination, soak the mouse body in 70% (v/v) ethanol before removing the femurs. Use instruments disinfected in 70% ethanol for the dissection. Collect the two femurs and clean them by removing any adherent tissue. After rapid immersion in ethanol, place them in 15 mL centrifuge tube containing 2 mL Tyrode&#39;s buffer.</li>
	<li>Cut away the epiphyses using a sharp razor blade and flush the bone marrow using a 5 mL syringe filled with 2 mL Tyrdode&#39;s buffer. To achieve this, introduce a 21 G needle into the opening of the femur (on the knee side) and slowly press the plunger to retrieve an intact marrow cylinder (Figure 1B,C). Keep the marrow collection session as brief as possible (under 10 min).</li>
</ol>
4. Bone marrow sectioning and placement into the incubation chamber
<ol>
	<li>Use a 3 mL plastic pipette to carefully and gently transfer the intact bone marrow onto a glass slide. It is important that the samples are covered with buffer to prevent drying out (Figure 1D). 
	NOTE: Avoid flow-reflow to minimize shears that could dissociate the tissue.</li>
	<li>Under a stereomicroscope (10x), cut off the ends of the marrow that may have been compressed at the time of the flush. Then cut transversal sections with a sharp razor blade. Sections should be thin enough to allow a detailed observation but ensure that megakaryocytes are not damaged by compression (usually thickness around 0.5 mm) (Figure 1E,F). 
	NOTE: The sections are made with a sharp razor blade and under a magnifier to adjust their thickness to about 0.5 mm. Only the sections of uniform thickness are selected. This procedure is not complicated but its standardization requires some experience.</li>
	<li>Using a plastic pipette, collect 10 sections into 1 mL tube containing Tyrode&#39;s buffer (Figure 1G).</li>
	<li>Carefully transfer the sections to an incubation chamber with a diameter of 13 mm (Figure 1H).</li>
	<li>Aspirate the buffer and adjust the volume to 30 &#181;L of Tyrode&#39;s buffer supplemented with 5 % mouse serum. 
	NOTE: It is at this stage that pharmacological agents can be added to evaluate their impact on proplatelet formation.</li>
	<li>Position the sections at distance. Seal the self-adhesive chamber with a coverslip of the dimension 22 x 55 mm. Incline the coverslip while sticking to avoid the formation of air bubbles (Figure 1I).</li>
	<li>Place the chamber in the heating chamber at 37 &#176;C. From this moment, the chronometer is started (T= 0 h). The experiment runs for 6 h at 37 &#176;C (T= 6 h).</li>
</ol>
5. Real-time observation of marrow explants
<ol>
	<li>Use an inverted phase contrast microscope (40x lens for magnification) coupled to a video camera to observe the bone marrow explants. Let the cells incubate for 30 min before starting the observation. While observing, it is necessary to adjust the focus because megakaryocytes are moving. 
	NOTE: Other observation modes are possible (e.g., DIC, fluorescence using mice whose megakaryocytes express an endogenous fluorescent marker), but phase contrast microscopy is optimal to clearly visualize the long and thin megakaryocyte extensions, allowing precise quantification. After 30 min, the marrow cells gradually migrate to the periphery of the explant, forming a monolayer. After 1 h of incubation, megakaryocytes can be identified by their large size and polylobulated nuclei (Figure 1J,K). After 3 h of incubation, the number of megakaryocytes increases and some have long extensions.</li>
	<li>Make videos to record the transformation of the megakaryocytes.</li>
</ol>
6. Quantification of the proplatelet-extending megakaryocytes
<ol>
	<li>Draw a map to localize each section in the incubation chamber (Figure 1K).</li>
	<li>After 1 h, identify the visible megakaryocytes (i.e., giant polylobulated cells) on each section&#39;s periphery and plot their positions on the drawing. Repeat this procedure after 3 and 6 h. 
	NOTE: On the basis of the drawing, each megakaryocyte can be easily located over time and the evolution of their morphology analyzed (e.g., size, deformation, proplatelet extension, etc.). Another possibility is the use of a specific navigation software.</li>
</ol>

<ol><li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+platelet+genesis%3B+in+vitro+study+by+cinemicrophotography%5BTitle%5D)+AND+%22Reviews+in+Hematology%22%5BJournal%5D)">Mechanism of platelet genesis; in vitro study by cinemicrophotography.</a> Reviews in Hematology. 11 (2), 162-174 (1956).</li>
<li>Becker, R. P., De Bruyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+transmural+passage+of+blood+cells+into+myeloid+sinusoids+and+the+entry+of+platelets+into+the+sinusoidal+circulation%3A+A+scanning+electron+microscopic+investigation%5BTitle%5D)+AND+%22American+Journal+of+Anatomy%22%5BJournal%5D)">The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation: A scanning electron microscopic investigation.</a> American Journal of Anatomy. 145 (2), 183-205 (1976).</li>
<li>Muto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+scanning+and+transmission+electron+microscopic+study+on+rat+bone+marrow+sinuses+and+transmural+migration+of+blood+cells%5BTitle%5D)+AND+%22Archivum+Histologicum+Japonicum%22%5BJournal%5D)">A scanning and transmission electron microscopic study on rat bone marrow sinuses and transmural migration of blood cells.</a> Archivum Histologicum Japonicum. 39 (1), 51-66 (1976).</li>
<li>Cramer, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+of+platelet+formation+by+human+megakaryocytes+cultured+with+the+Mpl+ligand%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.</a> Blood. 89 (7), 2336-2346 (1997).</li>
<li>Kaushansky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promotion+of+megakaryocyte+progenitor+expansion+and+differentiation+by+the+c-Mpl+ligand+thrombopoietin%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.</a> Nature. 369 (6481), 568-571 (1994).</li>
<li>Strassel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li>
<li>Aguilar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128 (16), 2022-2032 (2016).</li>
<li>Scandola, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+electron+microscopy+to+study+megakaryocytes%5BTitle%5D)+AND+%22Platelets%22%5BJournal%5D)">Use of electron microscopy to study megakaryocytes.</a> Platelets. 31 (5), 589-598 (2020).</li>
<li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+megakaryocyte+development+in+the+native+bone+marrow+environment%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Characterization of megakaryocyte development in the native bone marrow environment.</a> Methods in Molecular Biology. 788, 175-192 (2012).</li>
<li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li>
<li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice%5BTitle%5D)+AND+%22Journal+of+Thrombosis+and+Haemostasis%22%5BJournal%5D)">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li>
<li>Ortiz-Rivero, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C3G%2C+through+its+GEF+activity%2C+induces+megakaryocytic+differentiation+and+proplatelet+formation%5BTitle%5D)+AND+%22Cell+Communication+and+Signaling%22%5BJournal%5D)">C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.</a> Cell Communication and Signaling. 16 (1), 101(2018).</li>
<li>Junt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+visualization+of+thrombopoiesis+within+bone+marrow%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317 (5845), 1767-1770 (2007).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+MYH9-related+disease%3A+mutations+in+nonmuscle+myosin+II-A%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li>
<li>Malara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li>
<li>Balduini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adhesive+receptors%2C+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes%5BTitle%5D)+AND+%22Journal+of+Thrombosis+and+Haemostasis%22%5BJournal%5D)">Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.</a> Journal of Thrombosis and Haemostasis. 6 (11), 1900-1907 (2008).</li>
</ol>

The authors declare no conflicts of interests.

Here we describe a simple and low-cost in vitro method to evaluate the efficiency of megakaryocytes to extend proplatelets which have grown in the bone marrow. The bone marrow explant model for mouse has four main advantages. First, there are no advanced technical skills required. Second, the time needed to obtain megakaryocyte-extending proplatelets is quite short, only 6 hours for the explant method, compared to a minimum of 4 days for a conventional culture method starting from mouse progenitors. Third, given...

The bone marrow explant technique was first developed by Thi&#233;ry and Bessis in 1956 to describe the formation of rat megakaryocyte cytoplasmic extensions as an initial event in platelet formation1. Using phase contrast and cinematographic techniques, these authors characterized the transformation of mature round megakaryocytes into &#34;squid-like&#34; thrombocytogenic cells with cytoplasmic extensions showing dynamic movements of elongation and contraction. These arms become progressively thinner until they become filiform with small swellings along the arms and at the tips. These typical megakaryocyte elongations, obtained in vitro and in liquid media, have certain similarities with platelets observed in fixed bone marrow, where megakaryocytes protrude long extensions through the sinusoid walls into the blood circulation2,3. The discovery and cloning of TPO in 1994, allowed to differentiate megakaryocytes in culture able to form proplatelet extensions resembling those described in bone marrow explants4,5,6. However, megakaryocyte maturation is far less efficient in culture conditions, notably the extensive internal membrane network of bone marrow matured megakaryocytes is underdeveloped in cultured megakaryocytes, hampering studies on the mechanisms of platelet biogenesis7,8.
We detail here the bone marrow explant model, based on Thi&#233;ry and Bessis, to follow in real-time proplatelet formation of mouse megakaryocytes, which have fully matured in their native environment, thus circumventing possible in vitro artifacts and misinterpretations. Results obtained in wild-type adult mice are presented to illustrate the ability of megakaryocyte to extend proplatelets, their morphology and the complexity of proplatelets. We also introduce a rapid quantifying strategy for quality validation to ensure data accuracy and robustness during the megakaryocyte recording process. The protocol presented here is the most recent version of the method&#160;published as a book chapter previously9.

<table><tbody><tr><td>5 mL syringes</td><td>Terumo</td><td>SS+05S1</td><td></td></tr><tr><td>21-gauge needles</td><td>BD Microlance</td><td>301155</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>21108</td><td></td></tr><tr><td>Coverwall Incubation Chambers</td><td>Electron Microscopy Sciences</td><td>70324-02</td><td>Depth : 0,2 mm</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H-3375</td><td>pH adjusted to 7.5</td></tr><tr><td>Human serum albumin</td><td>VIALEBEX</td><td>authorized medication : n&amp;deg; 3400956446995</td><td>20% (200mg/mL -100mL)</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>BVBW8448</td><td></td></tr><tr><td>Micro Cover Glass</td><td>Electron Microscopy Sciences</td><td>72200-40</td><td>22 mm x 55 mm</td></tr><tr><td>Microscope</td><td>Leica Microsystems SA, Westlar, Germany</td><td>DMI8 - 514341</td><td>air lens</td></tr><tr><td>microscope camera</td><td>Leica Microsystems SA, Westlar, Germany</td><td>K5 CMS GmbH -14401137</td><td>image resolution : 4.2 megapixel</td></tr><tr><td>Mouse serum</td><td>BioWest</td><td>S2160-010</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>PSG 100x</td><td>Gibco, Life Technologies</td><td>1037-016</td><td>10,000 units/mL penicillin, 10,000 &amp;mu;g/mL streptomycin and 29.2 mg/mL glutamine</td></tr><tr><td>Razor blade</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Sucrose D (+)</td><td>Sigma</td><td>G8270</td><td></td></tr></tbody></table>

proplatelet formation dynamics of mouse fresh bone marrow explants

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.

Qualitative results. At the beginning of the experiment, all cells are compacted in the bone marrow section. It takes 30 min for the cells to become clearly visible at the periphery of the explants. The megakaryocytes are then recognizable by their large size and their evolution can then be studied over time (size, shape, dynamic, proplatelet extension and platelet release) (Figure 2A). Small megakaryocytes have a diameter between 20 and 30 µm and their nuclei are polylobula...

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 ...

proplatelet-formation-dynamics-of-mouse-fresh-bone-marrow-explants

Research

JoVE Journal

Biology

This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells — isolated from human umbilical cord blood — differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

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Learn a rapid explant method to visualize proplatelet formation from mouse megakaryocytes in native bone marrow. This protocol enables real-time observation and quantitative analysis of megakaryocyte behavior for hematology research.

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

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