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Biology

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Published: May 20th, 2021

DOI:

10.3791/62501

1Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

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 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 Thiéry 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 °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.

The bone marrow explant technique was first developed by Thiéry 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 "squid-like" 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

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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 (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg).

1. Preparation of reagents

  1. Prepare reagents as described in Table 1.
    1. For the Stock I, dissolve each powder separately. Ensure that the osmolarity of the preparation is hig.......

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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 polyl.......

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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.......

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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.

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Name Company Catalog Number Comments
5 mL syringes Terumo SS+05S1
21-gauge needles BD Microlance 301155
CaCl2.6H2O Sigma 21108
Coverwall Incubation Chambers Electron Microscopy Sciences 70324-02 Depth : 0,2 mm
HEPES Sigma H-3375 pH adjusted to 7.5
Human serum albumin VIALEBEX authorized medication : n° 3400956446995 20% (200mg/mL -100mL)
KCl Sigma P9333
MgCl2.6H2O Sigma BVBW8448
Micro Cover Glass Electron Microscopy Sciences 72200-40 22 mm x 55 mm
Microscope Leica Microsystems SA, Westlar, Germany DMI8 - 514341 air lens
microscope camera Leica Microsystems SA, Westlar, Germany K5 CMS GmbH -14401137 image resolution : 4.2 megapixel
Mouse serum BioWest S2160-010
NaCl Sigma S7653
NaH2PO4.H2O Sigma S9638
NaHCO3 Sigma S5761
PSG 100x Gibco, Life Technologies 1037-016 10,000 units/mL penicillin, 10,000 μg/mL streptomycin and 29.2 mg/mL glutamine
Razor blade Electron Microscopy Sciences 72000
Sucrose D (+) Sigma G8270

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