Name: proplatelet formation dynamics of mouse fresh bone marrow explants
Uploaded: 2021-05-20T15:00:00
Description: Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Die Autoren danken Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert für die technische Unterstützung. Diese Arbeit wurde von ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 und ANR-18-CE14-0037 unterstützt.

Alle Tierversuche wurden in Übereinstimmung mit den europäischen Normen 2010/63/EU und dem CREMEAS-Ausschuss für die Ethik von Tierversuchen der Universität Straßburg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg) durchgeführt.
1. Herstellung von Reagenzien
<ol><li>Reagenzien wie in Tabelle 1beschrieben vorbereiten.<ol>
<li>Für die Brühe I jedes Pulver separat auflösen. Stellen Sie sicher, dass die Osmolarität der Zubereitung höher...

<ol>
	<li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+platelet+genesis;+in+vitro+study+by+cinemicrophotography.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmural+passage+of+blood+cells+into+myeloid+sinusoids+and+the+entry+of+platelets+into+the+sinusoidal+circulation:+A+scanning+electron+microscopic+investigation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scanning+and+transmission+electron+microscopic+study+on+rat+bone+marrow+sinuses+and+transmural+migration+of+blood+cells.">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/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>Kaushansky, K., 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=Promotion+of+megakaryocyte+progenitor+expansion+and+differentiation+by+the+c-Mpl+ligand+thrombopoietin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">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/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. 31 (5), 589-598 (2020).</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> Methods in Molecular Biology. 788, 175-192 (2012).</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=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C3G,+through+its+GEF+activity,+induces+megakaryocytic+differentiation+and+proplatelet+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+receptors,+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes.">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>

Hier beschreiben wir eine einfache und kostengünstige In-vitro-Methode zur Bewertung der Effizienz von Megakaryozyten zur Erweiterung von Proplaten, die im Knochenmark gewachsen sind. Das Knochenmark-Explantat-Modell für Die Maus hat vier Hauptvorteile. Erstens sind keine fortgeschrittenen technischen Fähigkeiten erforderlich. Zweitens ist die Zeit, die benötigt wird, um Megakaryozyten-verlängernde Proplatelets zu erhalten, ziemlich kurz, nur 6 Stunden für die Explantat-Methode, verglichen mit einem Minimu...

Die Knochenmark-Explantationstechnik wurde erstmals 1956 von Thiéry und Bessis entwickelt, um die Bildung von zytoplasmatischen Erweiterungen von Megakaryozyten bei Ratten als erstes Ereignis bei der Thrombozytenbildung zu beschreiben1. Mit Hilfe von Phasenkontrast und kinematografischen Techniken charakterisierten diese Autoren die Umwandlung reifer runder Megakaryozyten in "tintenfischartige" thrombozytogene Zellen mit zytoplasmatischen Erweiterungen, die dynamische Bewegungen der Dehnung und Kontraktion zeigen. Diese Arme werden zunehmend dünner, bis sie mit kleinen Schwellungen entlang der Arme und an den Spitzen filiform werden. Diese typischen Megakaryozytendehnungen, die in vitro und in flüssigen Medien erhalten werden, weisen bestimmte Ähnlichkeiten mit Blutplättchen auf, die im festsitzenden Knochenmark beobachtet wurden, wo Megakaryozyten lange Ausdehnungen durch die Sinuswände in den Blutkreislauf ragen2,3. Die Entdeckung und Klonierung von TPO im Jahr 1994 ermöglichte es, Megakaryozyten in Kultur zu differenzieren, die in der Lage sind, Proplatleterweiterungen zu bilden, die denen ähneln, die in den Knochenmark-Explantaten4,5,6beschrieben sind. Die Megakaryozytenreifung ist jedoch unter Kulturbedingungen weit weniger effizient, insbesondere das ausgedehnte interne Membrannetzwerk von knochenmarkgereiften Megakaryozyten ist in kultivierten Megakaryozyten unterentwickelt, was Studien über die Mechanismen der Thrombozytenbiogenese behindert7,8.
Wir beschreiben hier das Knochenmark-Explantatmodell, das auf Thiéry und Bessis basiert, um in Echtzeit die Proplatletbildung von Maus-Megakaryozyten zu verfolgen, die in ihrer nativen Umgebung vollständig gereift sind und so mögliche In-vitro-Artefakte und Fehlinterpretationen umgehen. Ergebnisse, die an erwachsenen Wildtyp-Mäusen gewonnen wurden, werden vorgestellt, um die Fähigkeit von Megakaryozyten zur Erweiterung von Proplaten, ihre Morphologie und die Komplexität von Proplaten zu veranschaulichen. Wir führen auch eine schnelle Quantifizierungsstrategie für die Qualitätsvalidierung ein, um die Genauigkeit und Robustheit der Daten während des Megakaryozyten-Aufzeichnungsprozesses sicherzustellen. Das hier vorgestellte Protokoll ist die neueste Version der Methode, die als Buch kapitel zuvor9veröffentlicht wurde.

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	<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>CaCl2.6H2O</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&#176; 3400956446995</td>
			<td>20% (200mg/mL -100mL)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</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>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</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 &#956;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

Das letzte Stadium der Megakaryopoese führt zu zytoplasmatischen Erweiterungen aus reifen Megakaryozyten, den sogenannten Proplaten. Es wurde viel über die Proplatbildung unter Verwendung von in vitro-differenziertenMegakaryozyten gelernt; Es gibt jedoch immer mehr Hinweise darauf, dass herkömmliche Kultursysteme den Differenzierungs- / Reifungsprozess, der im Knochenmark stattfindet, nicht getreu rekapitulieren. In diesem Manuskript stellen wir eine explantierte Methode vor, die ursprünglich 1956 von Thiéry und Bessis beschrieben wurde, um Megakaryozyten zu visualisieren, die in ihrer natürlichen Umgebung gereift sind, wodurch potenzielle Artefakte und Fehlinterpretationen umgangen werden. Frische Knochenmarke werden durch Spülen der Oberschenkelknochen von Mäusen gesammelt, in 0,5 mm Querschnitte geschnitten und in eine Inkubationskammer bei 37 °C gegeben, die einen physiologischen Puffer enthält. Megakaryozyten werden allmählich an der explantaten Peripherie sichtbar und werden bis zu 6 Stunden unter einem inversen Mikroskop in Verbindung mit einer Videokamera beobachtet. Im Laufe der Zeit ändern Megakaryozyten ihre Form, wobei einige Zellen eine kugelförmige Form haben und andere dicke Erweiterungen entwickeln oder viele dünne Proplateletten mit ausgedehnter Verzweigung verlängern. Es werden sowohl qualitative als auch quantitative Untersuchungen durchgeführt. Diese Methode hat den Vorteil, dass sie einfach, reproduzierbar und schnell ist, da zahlreiche Megakaryozyten vorhanden sind, und klassischerweise bildet die Hälfte von ihnen Proplatelette in 6 Stunden im Vergleich zu 4 Tagen für kultivierte Maus-Megakaryozyten. Neben der Untersuchung von mutierten Mäusen ist eine interessante Anwendung dieser Methode die einfache Bewertung der pharmakologischen Wirkstoffe auf dem Proplatlet-Erweiterungsprozess, ohne den Differenzierungsprozess, der in Kulturen auftreten kann, zu stören.

Qualitative Ergebnisse. Zu Beginn des Experiments werden alle Zellen im Knochenmarkabschnitt verdichtet. Es dauert 30 Minuten, bis die Zellen an der Peripherie der Explantate deutlich sichtbar werden. Die Megakaryozyten sind dann an ihrer Größe erkennbar und ihre Entwicklung kann dann im Laufe der Zeit untersucht werden (Größe, Form, Dynamik, Proplatletverlängerung und Thrombozytenfreisetzung) (Abbildung 2A). Kleine Megakaryozyten haben einen Durchmesser zwischen 20 und...

Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Hier beschreiben wir die Knochenmark-Explantationsmethode, von der Probenvorbereitung bis zur mikroskopischen Objektträgeranalyse, um die Fähigkeit von Megakaryozyten, die sich in ihrer physiologischen Umgebung differenziert haben, zu bewerten, Proplatelette zu bilden.

Proplatbildungsdynamik von mausfrischen Knochenmark-Explantaten

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

Das übergeordnete Ziel dieses Verfahrens ist es, in Echtzeit die Bildung von Proplatelett aus Megakaryozyten der Maus zu verfolgen, die in ihrer natürlichen Umgebung gereift sind. Diese Methode hat den Vorteil, dass sie einfach, reproduzierbar und schnell ist. Man kann viele Megakaryozyten analysieren und die Bildung von Proplaten in nur sechs Stunden visualisieren, anstatt auf den ersten Tag der Kultur zu warten, wie bei Maus-Megakaryozyten in vitro.Eine interessante Anwendung dieser Methode ist die Möglichkeit, die Wirkung der pharmazeutischen Behandlung oder genetischen Mutation zu untersuchen, insbesondere im Schritt der Proplatatadierung. Hier gibt es keinen Eingriff in den Differenzierungsprozess, wie im Fall der In-vitro-Kultur. Dieses Video hilft, diese wichtigen Schritte des Spülens und Schneidens des Knochenmarks sicherzustellen.Es wird auch erklärt, wie man die Morphologie und Megakaryozyten klassifiziert, was für die Charakterisierung von Defekten bei thrombophilen Erkrankungen wirklich nützlich ist. Erwärmen Sie zunächst Tyrodes Puffer auf 37 Grad Celsius und schalten Sie die Heizkammer des Mikroskops ein, um die Temperatur auf 37 Grad Celsius zu bringen. Bereiten Sie alle notwendigen Werkzeuge wie einen Timer, Inkubationskammern, fünf Milliliter 21 Gauge Spritzen, Pinzette, eine Rasierklinge, Pasteur pipetten, Glasobjektträger und 15 Milliliter Zentrifugenröhrchen vor.Spülen Sie das Knochenmark mit zwei Millilitern Tyrodes Puffer, indem Sie eine 21-Gauge-Nadel in die Öffnung des Femurs auf der Knieseite einführen, und drücken Sie langsam auf den Kolben, um einen intakten Markzylinder zu entnehmen. Verwenden Sie eine Drei-Minuten-Liter-Kunststoffpipette, um das intakte Knochenmark vorsichtig und vorsichtig auf einen Glasträger zu übertragen. Schneiden Sie die Enden des Knochenmarks ab, die zum Zeitpunkt der Spülung möglicherweise komprimiert wurden.Schneiden Sie dann dünne Querschnitte von 0,5 Millimeter Dicke mit einer scharfen Rasierklinge, um eine Beschädigung der Megakaryozyten zu vermeiden. Sammeln Sie mit einer Kunststoffpipette 10 Abschnitte in einem Ein-Milliliter-Röhrchen, das Tyrodes Puffer enthält. Die Abschnitte werden vorsichtig in eine Inkubationskammer mit einem Durchmesser von 13 Millimetern überführt.Saugen Sie den Puffer ab und stellen Sie das Volumen auf 30 Mikroliter tyrodes Puffer ein, der mit 5% Mausserum ergänzt wird. Positionieren Sie die Abschnitte in einiger Entfernung. Versiegeln Sie die selbstklebende Kammer mit einem 22 x 55 Millimeter großen Abdeckschlupf und neigen Sie den Abdeckschlitten an, um die Bildung von Luftblasen zu vermeiden.Stellen Sie die Kammer bei 37 Grad Celsius in die Heizkammer. Starten Sie den Chronometer und führen Sie das Experiment sechs Stunden lang bei 37 Grad Celsius durch, Machen Sie Videos, um die Transformation der Megakaryozyten aufzuzeichnen. Nach 30 Minuten wandern die Markzellen allmählich an die Peripherie der Explantation und bilden eine Monoschicht.Nach einer Stunde Inkubation können Megakaryozyten anhand ihrer größe und polylobulierten Kerne identifiziert werden. Die Anzahl der Megakaryozyten nimmt nach drei Stunden Inkubation zu, und einige haben lange Verlängerungen. Zeichnen Sie eine Karte, um jeden Abschnitt in der Inkubationskammer zu lokalisieren.Identifizieren Sie nach einer Stunde die sichtbaren Megakaryozyten, bei denen es sich um riesige polylobulierte Zellen an der Peripherie jedes Abschnitts handelt, und zeichnen Sie ihre Positionen auf der Zeichnung auf. Wiederholen Sie diesen Vorgang nach drei und sechs Stunden. Megakaryozyten werden manuell gezählt und drei und sechs Stunden nach dem Versiegeln der Inkubationskammer nach ihrer Morphologie klassifiziert.Sie werden als klein, groß mit dicker Absicht und Proplatlet-Erweiterung klassifiziert. Mit Hilfe der Kartierung kann ihre Entwicklung im Laufe der Zeit verfolgt werden. Die Ergebnisse werden als Prozentsatz jeder Klasse zu jedem Beobachtungszeitpunkt ausgedrückt.Klassischerweise erstreckt sich die Hälfte der an der Peripherie sichtbaren Megakaryozyten nach sechs Stunden für das Knochenmark der Wildtyp-Maus. Dem Schicksal der runden Megakaryozyten folgte die Aufnahme sequenzieller Bilder im Laufe der Zeit, um abzubilden, wie sie Proplatelette bilden. Eine wichtige Sache, an die Sie sich erinnern sollten, wenn Sie dieses Verfahren versuchen, ist, die Explantate für die Dauer des Experiments bei 37 Grad Celsius zu halten.Eine andere Temperatur kann die Ergebnisse verändern. Interessanterweise kann das Explantatprotokoll nach introvertierten Mikroskopieexperimenten verwendet werden. Am Ende werden die Megakaryozyten in den Explantraten des Knochenmarks von Natur aus fluoreszierend sein, und ihr Verhalten kann leicht untersucht werden.Dies ermöglicht es, verschiedene Behandlungen an denselben Tieren zu kombinieren und so die Anzahl der Mäuse zu reduzieren. Zusammenfassend lässt sich sagen, dass die Explantatmethode schnell und einfach ist und viele Informationen über die Kapazität natürlicher Megakaryozyten zu externen Proplaten liefert. Die daraus resultierenden qualitativen und quantitativen Befunde sind der Schlüssel zu unserem Verständnis thrombophiler Erkrankungen.in einem physiologischen oder pathologischen Kontext.

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

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Kultur myeloide dendritische Zellen aus dem Knochenmark Vorstufen

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Forschung

Biologie

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Generierung von Knochenmark murinen dendritischen Zellen für den Einsatz in 2-Photonen-Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing von hämatopoetischen Zellen im Knochenmark

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoklasten Ableitung aus Knochenmark der Maus

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-innere Zelle Masse Injektion von embryonalen Stammzellen führt zu höheren Chimerism Raten

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Ausdruck von fluoreszierenden Fusionsproteinen in murinen Knochenmark gewonnenen dendritischen Zellen und Makrophagen

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolierung, Reinigung und Differenzierung von Osteoclast-Laufläufern aus der Rattenmarkierung

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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.

In Vivo Zwei-Photonen-Bildgebung von Megakaryozyten und Blutplättchen im Knochenmark des Mausschädels

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