The authors wish to thank Fabienne Proamer, Jean-Yves Rinckel, David Hoffmann, Monique Freund for technical assistance. This work was supported by ARMESA (Association de Recherche et D&#233;veloppement en M&#233;decine et Sant&#233; Publique), the European Union through the European Regional Development Fund (ERDF) and by Grant ANR-17-CE14-0001-01 to H.d.S.

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). The protocol is schematically shown in Figure 1.
1. Bone marrow collection and fixation (&#160;Figure 1A)
CAUTION: This procedure includes carcinogenic, mutagenic, and/or toxic substances and is performed under a chemical extraction hood. Wear appropriate protective equipment such as gloves and protections glasses.
<ol>
	<li>Prepare the fixative solution consisting of 2.5% glutaraldehyde in cacodylate buffer (see Supplementary File).</li>
	<li>Bone marrow collection
	<ol>
		<li>Use adult C57BL/6 mice of either sex 12-18 weeks of age. Euthanize the mice by CO2 asphyxiation and cervical dislocation.</li>
		<li>With a pair of thin scissors, cut the skin around the thigh and use tweezers to peel the skin off. Remove the extremity of the paw and then cut between the hip and thigh. Detach tibia from femur by cutting at the knee articulation and remove adherent tissue on tibias and femurs by using a scalpel.</li>
		<li>Remove the epiphyses with a sharp razor blade. While holding the femur with tweezers, use a 5 mL syringe filled with cacodylate buffer with a 21 G needle to flush the bone marrow into a 15 mL tube filled with 2 mL cacodylate buffer. To do so, insert the bevel of the needle into the bone marrow opening and slowly press the plunger until the marrow is expelled.</li>
	</ol>
	</li>
	<li>Bone marrow fixation by rapid immersion into fixative.
	<ol>
		<li>Immediately after flushing, use a plastic pipette to transfer the bone marrow cylinder into 1 mL of fresh glutaraldehyde fixative solution (previously prepared in 1.1) for 60 min at room temperature. 
		&#8203;NOTE: To preserve the tissue, ensure that the entire process, from bone dissection to the fixation step, is completed in less than 10 min. For the fixation, ensure that the fixative solution is at room temperature to avoid heat shock.</li>
	</ol>
	</li>
</ol>
2. Embedding bone marrow in agarose
NOTE: Marrow tissue is not sufficiently cohesive to maintain its integrity during the different washing steps and material can be easily lost. To overcome this problem, the marrow is covered in a gel of agar before dehydration.
<ol>
	<li>Prepare the agarose solution as described in the Supplementary File.</li>
	<li>Wash the fixed marrow from section 1.3 in cacodylate buffer and transfer it carefully to a glass slide using a plastic pipette. Using a warm pipette, quickly apply a drop of 2% liquid agar to the bone marrow cylinder. 
	&#8203;NOTE: The agar solidifies quickly while cooling. To ensure a homogenous covering of the bone marrow, the agar solution has to be kept warm until it is deposited onto the slide.</li>
	<li>Quickly place the slide rapidly on ice until the agar solidifies (1-2 min).</li>
	<li>Under a microscope, use a sharp razor blade to cut and discard the extremities of the bone marrow cylinder because of possible tissue compression in these areas. Transfer the marrow blocks in 1.5 mL microcentrifuge tubes containing 1 mL of cacodylate buffer.</li>
</ol>
3. Embedding bone marrow in resin
CAUTION: Resin components are toxic; some are carcinogenic and must be handled with care under a chemical extraction hood. Use appropriate protective equipment such as gloves and protection glasses. Osmium tetroxide is highly volatile at room temperature and its vapors are very harmful to the eyes, nose, and throat. Before being discarded, 2% osmium tetroxide must be neutralized by adding twice its volume of vegetable oil.
<ol>
	<li>Prepare the epoxy resin as described in the Supplementary File.</li>
	<li>Resin embedding 
	NOTE: Keep the samples in the same microcentrifuge tubes during incubations in successive baths of osmium, uranyl acetate and ethanol. Aspirate the supernatants with a Pasteur pipette. The volume of solution used for each bath must equal at least 10x the volume of the sample.
	<ol>
		<li>Post-fix the blocks with 1% osmium in cacodylate buffer for 1 h at 4 &#176;C, wash once in cacodylate buffer and then once in distilled water.</li>
		<li>Stain with 4% uranyl acetate in distilled water for 1 h, wash twice in distilled water.</li>
		<li>Dehydrate through a graded series of ethanol in distilled water: 4 times in 75% ethanol for 5 min, followed by 3 times in 95% ethanol for 20 min and then 3 times in 100% ethanol for 20 min. At this step, take one syringe of epoxy resin out from the freezer. 
		NOTE: The protocol can be paused in 100% ethanol for 1 h.</li>
		<li>To obtain uniform infiltration and polymerization of epoxy resin inside the marrow, incubate first the blocks in 2 successive baths of propylene oxide for 15 min.</li>
		<li>Add a 1:1 mixture of 100% propylene oxide and epoxy resin and incubate for 1 h. Place the samples on a slow rotary shaker at room temperature.</li>
		<li>Add 100% epoxy resin leave the sample for overnight incubation under agitation.</li>
		<li>Add 100% epoxy resin for 2 h incubation, still under agitation.</li>
		<li>Under a microscope, place the marrow blocks into flat silicone molds. Orientate samples to permit subsequent transversal sectioning of the entire bone marrow. Fill the molds with epoxy resin and place them at 60 &#176;C for 48 h. 
		&#8203;NOTE: All solutions (except ethanol and propylene oxide) are filtered through 0.22 &#181;m filter to avoid samples contamination. To ensure adequate polymerization of the resin, avoid bubbles while filling the molds.</li>
	</ol>
	</li>
</ol>
4. Ultrathin sectioning (Figure 1B)
NOTE: Transmission EM requires thin tissue sections through which electrons can pass generating a projection image of the interior of cells, structure, and organization of inner organelles (granules, endoplasmic reticulum, Golgi) and the arrangement of intracellular cell membranes.
<ol>
	<li>Mount the sample block in an ultra-microtome support. Put it on the sample holder. Trim the samples at 45&#730; in order to remove the excess of resin around the tissue with a rotating diamond or tungsten milling cutter.</li>
	<li>Mount the samples on the ultramicrotome with a diamond knife blade equipped with a water tank. Cut transverse sections of 500 nm and 100 nm thickness for histological and TEM analyses, respectively. Collect floating sections on the water-surface with a loop.</li>
	<li>Deposit the 500 nm thick section on a glass slide and 100 nm thick sections on 200 mesh thin-bar copper grids with a paper filter underneath. Prepare five grids for one condition: stain two grids first and keep the three remaining grids as a backup if necessary.</li>
</ol>
5. Toluidine blue staining for histology
NOTE: Staining sections for histology is important for three reasons: 1) to make sure that the tissue is actually cut and not the resin, 2) to check the quality of the inclusion, and 3) to rapidly evaluate the marrow sample. If this is not correct, cut deeper in the block.
<ol>
	<li>Dry the semi-thin sections slide on a heat plate (60 &#176;C).</li>
	<li>Add filtered 1% toluidine blue/1 % sodium borate in distilled water on the slides and heat on a hot plate (60 &#176;C) for 1-2 min. Wash the slides with distilled water and let it dry on the heat plate.</li>
	<li>Mount sections on coverslips with a drop of Poly(butyl methacrylate-co-methyl methacrylate) mounting medium and examine under a light microscope.</li>
</ol>
6. Heavy metal staining for TEM observation (Figure 1C)
NOTE: For the contrast, the upper side of the grids are inverted on 100 &#181;L drops of each successive bath with a loop. Prior to use, each solution is 0.22 &#181;m filtered. Remove the excess of liquid between each bath by gently contact the grid side on a filter paper.
<ol>
	<li>Stain with 4% uranyl acetate in distilled water for 5 min.</li>
	<li>Wash 3 times in distilled water for 5 min.</li>
	<li>Stain with lead citrate for 3 min.</li>
	<li>Wash 3 times in distilled water for 5 min.</li>
	<li>Deposit the grids by the lower side in contact with the filter paper to let them dry. 
	&#8203;NOTE: Heavy metals react in the presence of carbon dioxide. To minimize the precipitates, avoid air displacement during the contrast, do not speak, keep the environment calm and turn off the air-conditioning.</li>
</ol>
7. TEM (Figure 1E)
NOTE: The sections are introduced in a TEM microscope and examined at 120 kV.
<ol>
	<li>First examine the sections at low magnification (&#60; 500x) to appreciate the general aspect of the preparation (absence of hole in the resin, folds/compression in the sections, precipitates due to staining).</li>
	<li>Then examine the sections at higher magnification (~ 2000x allowing to distinguish the stage of maturation). Count manually the megakaryocytes from each stage of maturation over whole transversal sections (see Representative Results on how do visually identify each stage). 
	NOTE: Each square of the grids is defined as an area for examination (which equals 16000 &#181;m2 for 200 mesh copper grids).</li>
	<li>To assess the number of megakaryocytes, quantify only the squares that are fully covered with a section. To do so, use a model based on the screening of ranges. Observe a first range of squares from an extremity of the section to another, then another range in the same way, etc. Using this procedure, screen fully and systematically the whole marrow transversal section square by square.</li>
	<li>For each square, score the number of Stage I, II or III megakaryocytes. 
	NOTE: Higher magnifications are required to analyze the granules, the DMS organization, the size of cytoplasmic territories and the polylobulated nucleus.</li>
</ol>

<ol>
	<li>Machlus, K. R., Italiano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incredible+journey:+From+megakaryocyte+development+to+platelet+formation.">The incredible journey: From megakaryocyte development to platelet formation.</a> The Journal of Cell Biology. 201 (6), 785-796 (2013).</li><li>Zucker-Franklin, D., Termin, C. S., Cooper, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+the+megakaryocytes+of+patients+infected+with+the+human+immune+deficiency+virus+(HIV-1).">Structural changes in the megakaryocytes of patients infected with the human immune deficiency virus (HIV-1).</a> American Journal of Pathology. 134 (6), 9 (1989).</li><li>Eckly, 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=Biogenesis+of+the+demarcation+membrane+system+(DMS)+in+megakaryocytes.">Biogenesis of the demarcation membrane system (DMS) in megakaryocytes.</a> Blood. 123 (6), 921-930 (2014).</li><li>Scandola, 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=Use+of+electron+microscopy+to+study+megakaryocytes.">Use of electron microscopy to study megakaryocytes.</a> Platelets. , 1-10 (2020).</li><li>Behnke, O., Forer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+megakaryocytes+to+platelets:+platelet+morphogenesis+takes+place+in+the+bloodstream.">From megakaryocytes to platelets: platelet morphogenesis takes place in the bloodstream.</a> European Journal of Haematology. 60, 3-23 (2009).</li><li>Eckly, 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=Characterization+of+megakaryocyte+development+in+the+native+bone+marrow+environment.">Characterization of megakaryocyte development in the native bone marrow environment.</a> Platelets and Megakaryocytes. 788, 175-192 (2012).</li><li>Brown, E., Carlin, L. M., Nerlov, C., Lo Celso, C., Poole, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+membrane+extrusion+sites+drive+megakaryocyte+migration+into+bone+marrow+blood+vessels.">Multiple membrane extrusion sites drive megakaryocyte migration into bone marrow blood vessels.</a> Life Science Alliance. 1 (2), 201800061 (2018).</li><li>Eckly, 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=Megakaryocytes+use+in+vivo+podosome-like+structures+working+collectively+to+penetrate+the+endothelial+barrier+of+bone+marrow+sinusoids.">Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.</a> Journal of Thrombosis and Haemostasis. , 15024 (2020).</li><li>Cramer, E. 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=Ultrastructure+of+platelet+formation+by+human+megakaryocytes+cultured+with+the+Mpl+ligand.">Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.</a> Blood. 89 (7), 2336-2346 (1997).</li><li>Heijnen, H. F. G., Debili, N., Vainchencker, W., Breton-Gorius, J., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivesicular+Bodies+Are+an+Intermediate+Stage+in+the+Formation+of+Platelet+&#945;-Granules.">Multivesicular Bodies Are an Intermediate Stage in the Formation of Platelet &#945;-Granules.</a> Blood. 7 (7), 2313-2325 (1998).</li><li>Gupta, N., Jadhav, K., Shah, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emperipolesis,+entosis+and+cell+cannibalism:+Demystifying+the+cloud.">Emperipolesis, entosis and cell cannibalism: Demystifying the cloud.</a> Journal of Oral and Maxillofacial Pathology. 21 (1), 92 (2017).</li><li>Centurione, L., 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=Increased+and+pathologic+emperipolesis+of+neutrophils+within+megakaryocytes+associated+with+marrow+fibrosis+in+GATA-1low+mice.">Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1low mice.</a> Blood. 104 (12), 3573-3580 (2004).</li><li>Ellis, S. L., 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=The+relationship+between+bone,+hemopoietic+stem+cells,+and+vasculature.">The relationship between bone, hemopoietic stem cells, and vasculature.</a> Blood. 118 (6), 1516-1524 (2011).</li><li>Bornert, 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=Cytoskeletal-based+mechanisms+differently+regulate+in+vivo+and+in+vitro+proplatelet+formation.">Cytoskeletal-based mechanisms differently regulate in vivo and in vitro proplatelet formation.</a> Haematologica. , (2020).</li></ol>

The authors have no conflicts of interests to declare.

Direct examination of megakaryocytes in their native environment is essential to understand megakaryopoiesis and platelet formation. In this manuscript, we provide a transmission electron microscopy method combining bone marrow flushing and fixation by immersion, allowing to dissect in situ the morphology characteristics of the entire process of megakaryocyte morphogenesis taking place in the bone marrow.
The flushing of the bone marrow is a critical step of this method, as the succes...

Megakaryocytes are specialized large polyploid cells, localized in the bone marrow, responsible for platelet production1. They originate from hematopoietic stem cells through an intricate maturation process, during which megakaryocyte precursors progressively increase in size, while undergoing extensive concomitant morphologic changes in the cytoplasm and nucleus2. During maturation, megakaryocytes develop a number of distinguishable structural elements including: a polylobulated nucleus, invaginations of the surface membrane that form the demarcation membrane system (DMS), a peripheral zone devoid of organelles surrounded by the actin based cytoskeletal network, and numerous organelles including &#945;-granules, dense granules, lysosomes, and multiple Golgi complexes. At the ultrastructural level, a major modification observed is the cytoplasmic compartmentalization into discrete regions delimited by the DMS3. This extensive supply of membranes will fuel the extension of long cytoplasmic processes in the initial phase of platelet production, which will then remodel into platelets inside the circulation. Any defect during megakaryocyte differentiation and maturation can affect platelet production in term of platelet count and/or platelet function. 
Thin layer transmission electron microscopy (TEM) has been the imaging approach of choice for decades providing high-quality ultrastructure of megakaryocytes that have shaped our understanding of the physiology of thrombopoiesis4,5. This paper focuses on a standardized TEM method allowing to capture the process of platelet biogenesis occurring in situ within the native bone marrow microenvironment, which could also serve as a basis to analyze any bone marrow cell type. We provide ultrastructural examples of the development of megakaryocytes from immature to fully mature, which extend cytoplasmic processes into the microcirculation of sinusoids6. We also describe an easy procedure to quantify the different megakaryocyte maturation stages, instructing on the regeneration and platelet production capacity of the bone marrow.

<table><tbody><tr><td>2,4,6-Tri(dimethylaminomethyl)phenol (DMP-30)</td><td>Ladd Research Industries, USA</td><td>21310</td><td></td></tr><tr><td>Agarose type LM-3 Low Melting Point Agar</td><td>Electron Microscopy Sciences, USA</td><td>1670-B</td><td></td></tr><tr><td>CaCl2 Calcium chloride hexahydrate</td><td>Merck, Germany</td><td>2083</td><td></td></tr><tr><td>Copper grids 200 mesh thin-bar</td><td>Oxford Instrument, Agar Scientifics, England</td><td>T200-CU</td><td></td></tr><tr><td>Dimethylarsinic acid sodium salt trihydrate</td><td>Merck, Germany</td><td>8.20670.0250</td><td></td></tr><tr><td>Dodecenyl Succinic Anhydride (DDSA)</td><td>Ladd Research Industries, USA</td><td>21340</td><td></td></tr><tr><td>Double Edge Stainless Razor blade</td><td>Electron Microscopy Sciences-EMS, USA</td><td>EM-72000</td><td></td></tr><tr><td>Ethanol absolut</td><td>VWR International, France</td><td>20821296</td><td></td></tr><tr><td>Filter paper, 90 mm diameter</td><td>Whatman, England</td><td>512-0326</td><td></td></tr><tr><td>Flat embedding silicone mould</td><td>Oxford Instrument, Agar Scientific, England</td><td>G3533</td><td></td></tr><tr><td>Glutaraldehyde 25%</td><td>Electron Microscopy Sciences-EMS, USA</td><td>16210</td><td></td></tr><tr><td>Heat plate Leica EMMP</td><td>Leica Microsystems GmbH, Austria</td><td>705402</td><td></td></tr><tr><td>Histo Diamond Knife 45&amp;deg;</td><td>Diatome, Switzerland</td><td>1044797</td><td></td></tr><tr><td>JEOL 2100 Plus TEM microscope</td><td>JEOL, Japan</td><td>EM-21001BU</td><td></td></tr><tr><td>Lead citrate - Ultrostain 2</td><td>Leica Microsystems GmbH, Austria</td><td>70 55 30 22</td><td></td></tr><tr><td>LX-112 resin</td><td>Ladd Research Industries, USA</td><td>21310</td><td></td></tr><tr><td>MgCl2 Magnesium chloride hexahydrate</td><td>Sigma, France</td><td>M2393-100g</td><td></td></tr><tr><td>Mounting medium - Poly(butyl methacrylate-co-methyl methacrylate)</td><td>Electron Microscopy Sciences-EMS, USA</td><td>15320</td><td></td></tr><tr><td>Nadic Methyl Anhydride (NMA)</td><td>Ladd Research Industries, USA</td><td>21350</td><td></td></tr><tr><td>Osmium tetroxide 2%</td><td>Merck, Germany</td><td>19172</td><td></td></tr><tr><td>Propylene oxide (1.2-epoxypropane)</td><td>Sigma, France</td><td>82320-250ML</td><td></td></tr><tr><td>Saline injectable solution 0.9% NaCl</td><td>C.D.M Lavoisier, France</td><td>MA 575 420 6</td><td></td></tr><tr><td>Scalpel Surgical steel blade</td><td>Swann-Morton, England</td><td>.0508</td><td></td></tr><tr><td>Sodium tetraborate - Borax</td><td>Sigma, France</td><td>B-9876</td><td></td></tr><tr><td>Sucrose</td><td>Merck, Germany</td><td>84100-1KG</td><td></td></tr><tr><td>Syringe filter 0.2&amp;micro;m</td><td>Pall Corporation, USA</td><td>514-4126</td><td></td></tr><tr><td>Toluidine blue</td><td>Ladd Research Industries, USA</td><td>N10-70975</td><td></td></tr><tr><td>Trimmer EM TRIM2</td><td>Leica Microsystems GmbH, Austria</td><td>702801</td><td></td></tr><tr><td>Ultramicrotome Ultracut UCT</td><td>Leica Microsystems GmbH, Austria</td><td>656201</td><td></td></tr><tr><td>Uranyl acetate</td><td>Ladd Research Industries, USA</td><td>23620</td><td></td></tr></tbody></table>

in situ exploration of murine megakaryopoiesis using transmission electron microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

Bone marrow histology 
Observation of the bone marrow toluidine blue histology under a light microscope is key to rapidly analyze the overall tissue architecture in terms of e.g., tissue compactness, microvessel continuity, and the size and shape of megakaryocytes (Figure 1D). It is performed before ultrathin sections to determine the need of cutting deeper in the bone marrow block. Due to their giant size and nuclear lobulation, the more mature megakar...

Watch this Scientific Journal Video about In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy at JoVE.com

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

in-situ-exploration-murine-megakaryopoiesis-using-transmission

Research

JoVE Journal

Biology

2.7K Views.  Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS. Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV. 

Video: In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Bioengineering

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Immunology and Infection

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

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.

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 ...

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 ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Summary

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Chapters in this video

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Tracking Mouse Bone Marrow Monocytes In Vivo

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34⁺ Cells

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Isolation of Mouse Megakaryocyte Progenitors

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells