Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Summary

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.

Abstract

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.

Introduction

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 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 circulation^2,3. 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 explants^4,5,6. 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 biogenesis^7,8.

We detail here the bone marrow explant model, based on Thiéry 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 published as a book chapter previously⁹.

Protocol

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 higher than 295 mOsm/L. This solution can be stored at 4 °C for one year.
   2. For the Tyrode'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 µm pores.

2. Preparation of the experimental set up

1. On the day of the experiment, warm the Tyrode's buffer at 37 °C, and turn on the heating chamber of the microscope to bring the temperature to 37 °C.
2. 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).

3. Isolation of the mouse bone marrow

1. 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.
2. 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's buffer.
3. Cut away the epiphyses using a sharp razor blade and flush the bone marrow using a 5 mL syringe filled with 2 mL Tyrdode'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).

4. Bone marrow sectioning and placement into the incubation chamber

1. 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.
2. 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.
3. Using a plastic pipette, collect 10 sections into 1 mL tube containing Tyrode's buffer (Figure 1G).
4. Carefully transfer the sections to an incubation chamber with a diameter of 13 mm (Figure 1H).
5. Aspirate the buffer and adjust the volume to 30 µL of Tyrode'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.
6. 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).
7. Place the chamber in the heating chamber at 37 °C. From this moment, the chronometer is started (T= 0 h). The experiment runs for 6 h at 37 °C (T= 6 h).

5. Real-time observation of marrow explants

1. 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.
2. Make videos to record the transformation of the megakaryocytes.

6. Quantification of the proplatelet-extending megakaryocytes

1. Draw a map to localize each section in the incubation chamber (Figure 1K).
2. After 1 h, identify the visible megakaryocytes (i.e., giant polylobulated cells) on each section'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.

Representative Results

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 polylobulated while mature round megakaryocytes are larger (> 30 µm in diameter) with an enlarged cytoplasm. A few dark megakaryocytes may be observed (Figure 2C). These represent dead cells whose proportion should not exceed 0.5%. A proportion higher than this value indicates a sample preparation problem. The morphology of the nucleus can be easily visualized by varying the focus.

Quantitative results. Megakaryocytes are counted manually as described in 6.2. and classified according to their morphology at 3 h and 6 h after sealing of the incubation chamber. Figure 2A summarizes the four essential megakaryocytes classes: (1) small MKs, (2) large MKs, (3) MKs with thick extensions, (4) MKs with thin, elongated, and ramified extensions. These later are the typical proplatelet-forming megakaryocytes, with the prominent characteristics of swellings along the proplatelets and the presence of refractive buds at their extremities. With the help of mapping (Figure 1K), their evolution can be followed over time. The results are expressed as a percentage of each class at each observation time. Classically, half of the megakaryocytes visible at the periphery extend proplatelets at 6 hours for wild-type C57BL/6 mouse bone marrow (Figure 2B).

It is possible to follow the fate of round MKs by capturing sequential images over time to image how they form proplatelets (Video 1). Interestingly, when MKs with thick extensions were monitored over a period of 3 h, it was observed that the thick extensions could either detach from the cell body and branch into proplatelets or retract to reform large round MKs.

	Reagents				H2O
Stock I*	16 g	0.4 g	2 g	0.116 g
	NaCl	KCL	NaHCO3	NaH2PO4,	to 100 mL
	(2.73 M)	(53.6 mM)	(238 mM)	(8.6 mM)
Stock II	2.033 g MgCl2.6H2O (0.1 M)
Stock III	2.19 g  CaCl2.6H2O (0.1 M)				to 100 mL
HEPES Stock**	119 g HEPES* (0.5M)				to 1 L
Tyrode’s Buffer***	5 mL Stock I	1 mL Stock II	2 mL Stock III	1 mL HEPES Stock	1.8 mL albumin Stock

Table 1: Preparation of the Tyrode's buffer. Each stock solution is indicated in the first column of the table. The composition as well as the amount of reagent (given in grams) required for each stock solution is indicated per row. The catalog number and the company of each reagent are given in the table of essential supplies.

Figure 1: Photographic representations of the sample preparation method for the bone marrow explant. (A) Experimental setup required for the bone marrow preparation. (B) A 21-gauge syringe-mounted needle is inserted into the bone. (C) The bone marrow is flushed into a tube containing Tyrode's buffer. (D) The bone marrow is then gently deposited on a glass slide. (E) The extremities of the bone marrow are cut off. (F) The marrow cylinder is cut into ten 0.5 mm thick sections. (G-H) The ten sections are transferred to an incubation chamber and observed at 37 °C using an inverted microscope. (I) Representative photo of an incubation chamber containing the ten sections of bone marrow. (J) Peripheral cells migrate to form a layer in which the megakaryocytes become visible. (K) Example of drawing illustrating the ten explant sections in the incubation chamber as well as the location of the megakaryocytes (X blue mark) that have migrated out of the tissue for each section. The arrow shows a megakaryocyte at the periphery. Please click here to view a larger version of this figure.

Figure 2: Morphological classification of megakaryocytes in explants over time. (A) Megakaryocytes are classified as "small", "large", with "thick extension" or "proplatelet-extending". Bars: 50 µm (B) Proportion of megakaryocytes in each class was determined at 1 h, 3 h and 6 h for a total of 1,468 megakaryocytes, showing that the proportion of "small" and "spherical" megakaryocytes decreases with time while, in parallel, the proportion of megakaryocytes extending proplatelets increases (n=6 mice). Typically, in the explants of a WT mouse after 3 h, between 8.3 and 11.5 megakaryocytes are observed per section. The error bar corresponds to the standard error of the mean for each sample. (C) Representative image of a dark megakaryocyte. Bar: 50 µm (D) Representative image of a megakaryocyte with non-muscular myosin II-A labeled with a green fluorescent protein. Bar: 50 µm Please click here to view a larger version of this figure.

Video 1: Time-lapse video showing a MK extending proplatelets. Please click here to download this Video.

Discussion

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 that only a small amount of tissue is needed and that the results obtained are reproducible, it reduces the number of mice needed to a minimum (usually 6 mice per experimental condition), making these experiments economically and ethically efficient. Lastly, but importantly, the strength of this method lies in the use of megakaryocytes that have fully developed in their natural environment, which may prove invaluable in revealing phenotypes that could be masked in vitro by potential artifacts of the culture conditions. This has been previously documented in mice with megakaryocyte-restricted MYH9 inactivation where opposing results have been found on proplatelet formation in in vitro (increased formation)¹⁰ and in vivo (decreased formation) differentiated megakaryocytes¹¹. These paradoxical results have been explained by the requirement of myosin IIA for normal megakaryocyte differentiation in a constraint environment, while myosin IIA is dispensable for megakaryocyte differentiation in liquid culture⁷.

An interesting application of the bone marrow explant model is the possibility to study the impact of genetic mutations or deficiencies in transgenic mice and/or pharmacological agents exclusively on the platelet extension process, without interfering with the differentiation process as in the case of in vitro culture¹². The ideal situation is to use the bone marrow of one femur as a treated sample and its counterpart as control. In addition, the use of transgenic mice allowing spontaneous fluorescence in the megakaryocytes facilitates the visualization of the platelet extension process. To visualize fluorescent megakaryocytes, one possibility could be to add fluorescence-labelled antibodies against specific megakaryocyte markers in the culture chamber. Another possibility could be the use of genetically engineered mouse models expressing a fluorescent protein, either specifically in the megakaryocytic lineage such as mice with CD41-labelled YFP already reported in the literature¹³, or in all cells such as mice where GFP is linked to the N-terminus of non-muscular myosin II-A¹⁴ as illustrated in Figure 2D.

This explant method, therefore, provides both qualitative and quantitative information for a better understanding of platelet formation in their natural environment. Noteworthy, although this method is simple and fast it remains complementary to the studies performed using classical liquid culture. Each one brings separate knowledge according to the stages of proliferation, maturation, extension of the platelets and platelet release that one wishes to study. For example, where the explant method gives information on the capacity of extension of proplatelets by megakaryocytes that have grown in a physiological context, in vitro culture provides information on the importance of the bone marrow microenvironment such as the impact of cell ridigity⁷ or extracellular matrix dependency¹⁵. Thus, in vitro megakaryocyte cultures make it possible to modulate the parameters of the microenvironment in terms of stiffness and adhesive proteins^7,16. Please refer to the article "Megakaryocyte culture in three-dimensional methylcellulose-based hydrogel to improve cell maturation and study the impact of stiffness and confinement" by J. Boscher et al., presented in this issue for more information.

Materials

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