In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 α-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 thrombopoiesis^4,5. 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.

Protokół

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). The protocol is schematically shown in Figure 1.

1. Bone marrow collection and fixation ( 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.

1. Prepare the fixative solution consisting of 2.5% glutaraldehyde in cacodylate buffer (see Supplementary File).
2. Bone marrow collection
   1. Use adult C57BL/6 mice of either sex 12-18 weeks of age. Euthanize the mice by CO₂ asphyxiation and cervical dislocation.
   2. 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.
   3. 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.
3. Bone marrow fixation by rapid immersion into fixative.
   1. 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.
      ​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.

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.

1. Prepare the agarose solution as described in the Supplementary File.
2. 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.
   ​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.
3. Quickly place the slide rapidly on ice until the agar solidifies (1-2 min).
4. 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.

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.

1. Prepare the epoxy resin as described in the Supplementary File.
2. 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.
   1. Post-fix the blocks with 1% osmium in cacodylate buffer for 1 h at 4 °C, wash once in cacodylate buffer and then once in distilled water.
   2. Stain with 4% uranyl acetate in distilled water for 1 h, wash twice in distilled water.
   3. 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.
   4. 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.
   5. 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.
   6. Add 100% epoxy resin leave the sample for overnight incubation under agitation.
   7. Add 100% epoxy resin for 2 h incubation, still under agitation.
   8. 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 °C for 48 h.
      ​NOTE: All solutions (except ethanol and propylene oxide) are filtered through 0.22 µm filter to avoid samples contamination. To ensure adequate polymerization of the resin, avoid bubbles while filling the molds.

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.

1. Mount the sample block in an ultra-microtome support. Put it on the sample holder. Trim the samples at 45˚ in order to remove the excess of resin around the tissue with a rotating diamond or tungsten milling cutter.
2. 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.
3. 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.

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.

1. Dry the semi-thin sections slide on a heat plate (60 °C).
2. Add filtered 1% toluidine blue/1 % sodium borate in distilled water on the slides and heat on a hot plate (60 °C) for 1-2 min. Wash the slides with distilled water and let it dry on the heat plate.
3. Mount sections on coverslips with a drop of Poly(butyl methacrylate-co-methyl methacrylate) mounting medium and examine under a light microscope.

6. Heavy metal staining for TEM observation (Figure 1C)

NOTE: For the contrast, the upper side of the grids are inverted on 100 µL drops of each successive bath with a loop. Prior to use, each solution is 0.22 µm filtered. Remove the excess of liquid between each bath by gently contact the grid side on a filter paper.

1. Stain with 4% uranyl acetate in distilled water for 5 min.
2. Wash 3 times in distilled water for 5 min.
3. Stain with lead citrate for 3 min.
4. Wash 3 times in distilled water for 5 min.
5. Deposit the grids by the lower side in contact with the filter paper to let them dry.
   ​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.

7. TEM (Figure 1E)

NOTE: The sections are introduced in a TEM microscope and examined at 120 kV.

1. First examine the sections at low magnification (< 500x) to appreciate the general aspect of the preparation (absence of hole in the resin, folds/compression in the sections, precipitates due to staining).
2. 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 µm2 for 200 mesh copper grids).
3. 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.
4. 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.

Wyniki

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...

Dyskusje

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...

Materiały

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