This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.

HINWEIS: Human Knochenmarkaspiraten aus dem Beckenkamm wurden vom mündigen Spender mit Zustimmung der Ethikkommission des Kantons Luzern gesammelt. Canine Knochenmarkaspiraten aus dem Beckenkamm wurden mit Hundebesitzers consent.Human gesammelt (ca. 20 ml) und Hunde (ca. 10 ml) Knochenmarkaspiraten waren antikoaguliert durch Zugabe von 15 ml 3,8% Natriumcitrat unmittelbar nach dem Rückzug im Operationssaal. Die Proben wurden auf die Laborumgebung zum Verarbeiten der gleiche Tag wie zurückführt. 1. Herstellung von Urokinase (Vor dem 1. Verwenden) <ol><li> Rekonstituieren Urokinase mit steriler phosphatgepufferter Salzlösung (PBS) nach den Anweisungen des Herstellers: 10 ml PBS zu dem Septum-Einlaß Kolben unter Verwendung einer 10 ml Spritze. Lösen Sie die Trockensubstanz durch Verwirbelung zu 50.000 Einspritzeinheiten (U) pro ml zu erhalten. </li><li> Bereiten Sie 500 ul Aliquots (jeweils 25.000 U) in sterile Rohre. Speicher Aliquots -20 ° C und verwenden es, wenn sie für mindestens 6 Monate benötigt. </li></ol> 2. Präparative Schritte (Vor jeder Knochenmarkbehandlung) <ol><li> Auftauen ein Aliquot von Urokinase (25.000 U) pro geronnen Knochenmarkaspirat (Aspirate Volumina bis zu 25 ml wurden erfolgreich unter Verwendung eines einzigen Aliquot 25.000 U behandelt wurde). </li><li> Heizen Sie ein Wasserbad oder Schüttelinkubator bis 37 ° C. </li></ol> 3. Enzymatische Digest des Clot <ol><li> Alle Arbeiten in einer laminaren Strömung Biosicherheitswerkbank, um eine mikrobielle Kontamination der Knochenmarkproben während der Verarbeitung zu vermeiden. Bei der Arbeit mit potenziell infektiöses humanes Material ist die Verwendung von Schutzhandschuhen sowie sorgfältige Umgang beraten. </li><li> Legen Sie eine 100 um Zellsieb auf einem sterilen 50 ml-Tube. Gießen Sie das Knochenmarkaspirat durch die Zelle Sieb, Kippen des Schmutzfänger oder bewegen das Gerinnungsmaterial. Verwenden Sie eine sterile Pipettenspitze fürbessere Durchströmung des Filtergitter. Hinweis: Vermeiden Sie jede Verdünnung des Gerinnsels, beispielsweise durch Waschen der Gerinnsel mit PBS. Urokinase wirkt indirekt und erfordert Komponenten des Serums (dh., Plasminogen) von der Biopsie zur wirksamen verdauen. Während der Fortsetzung des Verfahrens mit dem Gerinnungsmaterial, kann das Filtrat bei Raumtemperatur bis zur weiteren Verwendung in Schritt 3.6 gehalten werden. </li><li> Übertragen Sie die Knochenmark Blutgerinnsel Material in einen leeren Zellkulturschale mit einer sterilen Pinzette. Schneiden Sie den Schmutz in kleine Stücke von etwa 2 mm 3 mit einem sterilen Skalpell. </li><li> Übertragen der kleinen Stücke des Gerinnsels in einen 50 ml Reaktionsgefäß unter Verwendung der sterilen Pinzette. Stellen Sie sicher, dass das Hackfleisch Gerinnsel hat eine Wet-Optik. Wenn es trocken zu sein scheint, verwenden Sie etwas von dem Filtrat aus Schritt 3.1 zu befeuchten. </li><li> Man reibt das Gerinnsel durch Auf- und Abpipettieren für 5-mal mit einer 5 ml Pipette Mikrotiterplatten. 1 Aliquot von Urokinase an der Probe. Inkubieren für 30 min bei 37 ° C entweder in einem Wasserbad oder inein Schütteln (sanft) Inkubator. </li><li> Verreiben durch Auf- und Abpipettieren für 5 mal mit einer 5-ml-Pipette. Führen Sie diesen Schritt in einer Biosicherheitswerkbank. Inkubieren für weitere 30 min bei 37 ° C. Nach der Inkubation verreiben wieder 5-mal mit einer 5 ml Pipette. Fahren Sie über eine neue 100 um Zellsieb mit dem Filtrat aus Schritt 3.2 zu bündeln. </li><li> Zentrifuge die Zellsuspension bei Raumtemperatur für 10 min bei 500 x g. Überstand verwerfen. Re-suspend das Zellpellet in Medien wie unten oder nach dem Standardverfahren Ihres Labors für MSC Expansion Kultur beschrieben. </li></ol> 4. Seeding für Expansion Zellkultur und CFU-Platten <ol><li> Resuspendieren der Zellen (die aus Erythrozyten und mononukleäre Zellen) in 50 ml Basalmedium: Alpha-MEM mit 10% fötalem Rinderserum (FBS), 100 U / ml Penicillin, 100 mg / ml Streptomycin und 2,5 mg / ml Amphotericin B supplementiert . Zählen Sie die Zellen 1:10 in Trypanblau Lösung unter Verwendung einer Neubauer chamber. </li><li> Platte, die Zellen in Zellkulturflaschen in einer Dichte von 5 × 10 7 Zellen / cm 2 und Inkubation in einem befeuchteten Inkubator bei 37 ° C. Falls vorhanden, hypoxische (5% Sauerstoff) Bedingungen. Für den CFU-Assay Steuerung Platte 10 9 Zellen in einer Zellkulturschale von 10 cm Durchmesser. </li><li> Nach 3 Tagen Kultur werden die mesenchymalen Stammzellen an der Zellkulturschale befestigt ist, während andere Zellen in Suspension verbleiben. Ändern der Medien mit Basalmedium mit 5 ng / ml basischen Fibroblastenwachstumsfaktor (bFGF) ergänzt. Für die CFU-Test Kontrolle, Behandlung der Zellkulturschale ebenfalls. </li><li> Weiterhin Zellkultur für insgesamt 2 Wochen Austausch von Medien dreimal wöchentlich. Wenn die Zellen 80% Konfluenz überschreiten, durch Trypsinierung aufgeteilt. Für den CFU-Assay Steuerung, tauschen die Medien ebenfalls, doch spalten nicht die Zellen im Verlauf der zwei Wochen. </li></ol> 5. Giemsa Stain für CFU-Assay <ol><li> Bereiten Sie 10 ml Giemsa-Lösung pro Schale: dilute die Stammlösung (7,6 g / l Giemsa in Glycerin: Methanol) 1:10 in sterilem Wasser (immer eine neue Arbeitsverdünnung). </li><li> Druckmaterial aus der Zellkulturschale und waschen Sie die Zellen mit PBS. Seien Sie sehr vorsichtiger bei der Anwendung von Flüssigkeit auf die Petrischale und vermeiden Abwaschen der Zellen. </li><li> Fix Zellen in reinem Methanol für 5 min bei RT. Entsorgen Sie die Methanol. Hinzufügen die Giemsa-Lösung und Inkubation für 60 min in einem befeuchteten Inkubator bei 37 ° C. </li><li> Zweimal mit PBS waschen. Air zuerst trocknen Sie die Platte Kopf auf einem Papiertuch. Die Kolonien manuell. Dies wird am besten mit einem Filzstift auf der Rückseite der Platte erreicht. </li></ol> 6. MSC Differenzierung <ol><li> Canine MSCs Differenzierung HINWEIS: Canine MSCs wurden in chondrogene, knochenbildende und adipogene Linien durch Stimulation mit den entsprechenden Medien über vier Wochen differenziert. </li><li> Für Adipogene Differenzierung, Kultur MSCs in Monolayer bei 4 x 10 5 cells / cm 2 abwechselnd zwei unterschiedliche Kulturbedingungen wie folgt; <ol><li> Kultur in Adipogenese Erhaltungsmedium, das DMEM + GlutaMAX, 3% FBS, 100 Einheiten / ml Penicillin, 100 mg / ml Streptomycin, 2,5 ug / ml Amphotericin B und 170 mM Insulin. </li><li> Kultur in Adipogenese Induktionsmedium mit Erhaltungsmedium mit 3% FBS, 5% Kaninchenserum, 1 uM Dexamethason ergänzt, 500 uM 3-Isobutyl-1-Methylxanthin, 33 uM Biotin, 5 uM Rosiglitazon und 17 uM Pantothenat 16. </li><li> Schwelgen Sie die Lipidtröpfchen durch Färbung mit Oil Red-O. Kurz gesagt, reparieren Zellen mit 10% Formaldehyd, mit PBS waschen und färben mit 0,35% Oil red O in Isopropanol. </li></ol></li><li> Für osteogene Differenzierung Kultur MSCs in Monoschicht auf 7 x 10 3 Zellen / cm 2 und in der folgenden zu stimulieren: <ol><li> Erweiterte DMEM (GIBCO) + GlutaMAX, 5% FBS, 100 Einheiten / ml Penicillin, 100 mg / ml Streptomycin, 2,5 ug / ml Amphotericin B, 501; M L-Ascorbinsäure-2-phosphat, 10 mM ß-Glycerophosphat und 100 nM Dexamethason. </li><li> Identifizieren Sie die Mineralisierung Einlagen von Von Kossa-Färbung (5% AgNO 3). Kurz gesagt, fixieren Sie die Zellen mit 10% Formaldehyd, Waschen mit PBS und Fleck mit 5% AgNO 3 in destilliertem Wasser. </li></ol></li><li> Chondrogene Differenzierung <ol><li> Cut Würfel (3 mm je Seite) von einem Schwamm geformt Medizinprodukt, gebildet aus lyophilisierten Kollagen Typ I, und als Gerüstmaterial verwendet, um Zellen 17 zu unterstützen. </li><li> Seed MSCs auf der Oberseite der Würfel in einer Konzentration von 4 x 10 6 Zellen / ml (~ 70.000 Zellen / Cube). </li><li> Vor der Zugabe der Medien, um die Zellen, die den Würfel 30 Minuten einzuhalten. </li><li> Pflegen MSC-Kollagenstrukturen in chondrogenem Medium aus DMEM / F12 + GlutaMAX, 2,5% FBS, 100 Einheiten / ml Penicillin, 100 mg / ml Streptomycin, 2,5 ug / ml Amphotericin B, 40 ng / ml Dexamethason, 50 ug / ml Ascorbinsäure -2-phosphat,50 ug / ml L-Prolin, 1x Insulin-Transferrin-Selen-X, und 10 ng / ml transformierender Wachstumsfaktor-β1. </li><li> Verwenden Alcianblau Färbung, um die Ansammlung von Proteoglykanen in Konstrukte Abschnitte zu visualisieren. Kurz gesagt, färben die Abschnitte O / N mit 0,4% Alcianblau in 0,01% H 2 SO 4 und 0,5 M Guanidinhydrochlorid. Anschließend wurden die Schnitte waschen für 30 min in 40% DMSO und 0,05 M MgCl 2 gewaschen. Die Zellen wurden mit Kernechtrot gegengefärbt. </li></ol></li></ol>

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The authors have nothing to disclose.

Routinemäßig wir probieren Knochenmark, während der Patient in der Chirurgie (in unserem Fall vor allem Wirbelsäulenchirurgie), mit dem Vorteil, dass nur wenig zusätzliche Arbeit muss durch das Personal im Operationssaal durchgeführt werden. Auch wenn die Proben mit Natriumcitrat sofort nach Entnahme gemischt wurden viele Proben teilweise geronnen, wenn sie im Labor zur Verarbeitung angekommen. Zu diesem Zeitpunkt würde Resampling zu Clotted Exemplare zu ersetzen eine separate zusätzliche Intervention erforderli...

Mesenchymale Stammzellen (MSCs) spielen eine wichtige Rolle in der regenerativen Medizin und des Tissue Engineering. Sie können Migration, Differenzierung in verschiedene Zelltypen 1 und einprägen, die ihnen die idealen Kandidaten für autologe Therapien 2,3 macht. In letzter Zeit, klinischen Studien mit MSCs für Knochen- und Knorpelreparatur, Graft-versus-Host-Erkrankung oder Herzerkrankung wurden eingeführt 4. Diese MSCs aus der Nabelschnur oder Fettgewebe gewonnen werden, sondern die vielversprechendsten Ergebnisse wurden aus dem Knochenmark stammenden Stammzellen 5 erhalten. Der Beckenkamm ermöglicht, eine beträchtliche Menge von Knochenmark gesammelt und dient daher als Hauptstelle der Aspiration 6. Die Qualität des Aspirat nimmt jedoch mit zunehmendem Volumen von Knochenmark entnommen. Während die ersten 5 ml Knochenmarkaspirat enthält MSCs von hoher Qualität, Entzug von größeren Mengen führt zu einer Verwässerung des absaugen mit peripheren Blut fROM Die gefäßKnochen 7. Aufgrund der gegenwärtigen Megakaryozyten-Plättchen sind Knochenmarkaspiraten anfällig für die Blutgerinnung, es sei denn, Antikoagulanzien eingesetzt werden. Aber auch mit Antikoagulantien kann Blutgerinnsel auftreten. Im Knochenmark-MSCs stellen nur einen kleinen Anteil der Gesamtzellpool 8 und haben in Kultur für die meisten Gewebezüchtung oder therapeutische Anwendungen 4 erweitert werden. Die Qualität einer solchen Kultur hängt weitgehend von der ursprünglichen Zellpool, dh., Vielfalt und eine hohe Startnummer 9. Niedrige Werte von MSCs aus Entnahmen kann teilweise durch Spendervariabilität erklärt werden. Auf der anderen Seite, MSCs von niedriger Qualität Proben erfordern längere Zeit in der Kultur und erweitert Passage, um die gewünschte Anzahl von Zellen zu erreichen. In jedem Fall ist verlängert Passagierung eine Quelle von Zellalterung und kann zum Verlust der Differenzierungspotential 10 führen. Daher optimierte Protokolle, Zell y maximieren könnenield und verhindert nachteilige Effekte entwickelt 11,12 werden müssen. Als wir begannen, mit Hunde-MSCs zu arbeiten, waren wir erstaunt zu sehen, dass etwa drei von vier Hundeknochenmarkproben enthalten Blutgerinnsel, während glücklicherweise geronnen Humanproben (jede zehnte) wurden weniger häufig. Auf der anderen Seite war es keine Überraschung, dass wir viel niedrigeren Renditen von MSCs aus geronnenen Proben beobachtet. Um die wiederkehrende Frage der geronnenen Proben zu lösen, haben wir das Protokoll entwickelt, mit dem Thrombolytikum Urokinase anstelle von Resampling. Thrombolytische Therapien lebensbedrohlichen Situationen wie Verschluss von Blutgefäßen verursacht Herzinfarkt, Schlaganfall oder Embolien aufgrund unerwünschter Blutgerinnung entgegenwirken. Sie wirken, indem sie den Abbau der Thromben durch enzymatische Spaltung von Fibrin durch Plasmin und Enzym Plasminogenaktivatoren. Trotz der breiten Verwendung zur Behandlung von Patienten, existieren nur sehr wenige Veröffentlichungen, die verwendet thrombolytischen Aktivitätenfür Laboranwendungen bis geronnenen Proben zu retten, vor allem mit Schwerpunkt auf Lymphozyten. 1987 Nikus et al. beschrieben die Verwendung von Streptokinase zur Auflösung von Blutgerinnseln, was zu Funktions Lymphozyten 13 und vier Jahre später, De Vis et al. erweitert die Verwendung von Streptokinase Leukämiezellen aus Blut und Knochenmark für die Durchflusszytometrie-Anwendungen 14 zu isolieren. Eine neuere Veröffentlichung schlägt die Verwendung von Alteplase für die Krebsdiagnostik 15. Während der Verwendung des gleichen Enzym Ansatz konzentriert sich unser Protokoll für die Isolierung von multi MSCs Form Knochenmark, um ein Werkzeug für Forscher in der Stammzell-Bereich bieten.

<table border="0" cellpadding="0" cellspacing="0" width="674">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Basal Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PenStrep 100X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human FGF-basic</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MEM Alpha w/ Nucleoside, w/ stable Glutamine</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-23S50-I</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">FBS Heat Inactivated</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">2-01F36-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Amphotericin B</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1907</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Adipogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DMEM-HAM F12 + GlutaMAX</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-26F09-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rabbit serum&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">16120099</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Dexamethasone</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">D4902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5879</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biotin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">B4639</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rosiglitazone&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">R2408</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pantothenate&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5155</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oil Red-O&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">O0625</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Osteogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-ascorbic acid 2-phosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A8960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#223;-glycerophosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">G9422</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:328px;">Silver nitrate (AgNO3)</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">S6506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chondrogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biopad - sponge shaped medical device&#160;</td>
			<td style="width:227px;">Euroresearch</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-proline&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5607</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin-Transferrin-Selenium X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">51500056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human transforming growth factor-&#946;1&#160;</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Alcian Blue 8GX</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A3157</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Nuclear fast red</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">N8002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Generic</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tri-Sodium citrate dihydrate</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3901</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PBS</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">964.9100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Urokinase</td>
			<td style="width:227px;">Medac</td>
			<td style="width:119px;">1976826</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">0.5% Trypsin-EDTA</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15400054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Giemsa stain</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0885</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0877</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid (H2SO4)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0655</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1584</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidine hydrochloride</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1499</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Consumables</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">50 mL reaction tube</td>
			<td style="width:227px;">Axygen</td>
			<td style="width:119px;">SCT-50ML-25-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10 mL syringe</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4606108V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Sterican needle (22G)</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4657624</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">1.7 mL Microtubes</td>
			<td style="width:227px;">Brunschwig</td>
			<td style="width:119px;">MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">100 &#956;m cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">6.05935</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile forceps</td>
			<td style="width:227px;">Bastos Viegas, SA</td>
			<td style="width:119px;">489-001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile scalpel</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">5518059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Primaria cell cuture dish</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">353803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">C-Chip Neubauer Improved</td>
			<td style="width:227px;">Bioswisstech</td>
			<td style="width:119px;">505050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">cell culture flask - Flask T300</td>
			<td style="width:227px;">TPP</td>
			<td style="width:119px;">90301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Microbiological biosafety cabinet class II</td>
			<td style="width:227px;">Skan</td>
			<td style="width:119px;">82011500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">water bath</td>
			<td style="width:227px;">Memmert</td>
			<td style="width:119px;">1305.0377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Stripettes Serological Pipette 5ml</td>
			<td style="width:227px;">Corning</td>
			<td>4487-200ea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">microscope</td>
			<td style="width:227px;">Olympus</td>
			<td style="width:119px;">CKX41</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">humidified incubator Heracells 240</td>
			<td style="width:227px;">Thermo scientific</td>
			<td>51026331</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Heraeus Multifuge 1S-R</td>
			<td style="width:227px;">Thermo scientific</td>
			<td style="width:119px;">75004331</td>
		</tr>
	</tbody>
</table>

an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Die Tatsache, dass 74% der Hundeknochenmarkproben (n = 54) enthalten sind Blutgerinnsel, wenn sie in unserem Labor (1A) kam zusammen mit verringerter MSC Erträge aus diesen Proben, hat uns davon überzeugt, dass eine beträchtliche Anzahl von MSCs wurde innerhalb der Blutgerinnsel gefangen . Tatsächlich ist ein einfaches DAPI-Färbung des geschnittenen Nungsmaterial bestätigt die Anwesenheit von kernhaltigen Zellen in hoher Dichte (1B). Dies führt letztlich zu geringe Zahl von MSCs ...

Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Ein enzymatisches Verfahren zur mesenchymalen Stammzellen aus Knochenmarkproben Clotted Rettung

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

Research

JoVE Journal

Developmental Biology

9.1K Aufrufe.  Swiss Paraplegic Research. Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Serie: An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Isolierung von humanen mesenchymalen Stammzellen und deren Anbau an der porösen Knochenmatrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Forschung

Entwicklungsbiologie

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Epigenetische Umwandlung als eine sichere und einfache Methode, Insulin absondernden Zellen aus adulten Fibroblasten der Haut zu erhalten

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolierung und Expansion von mesenchymalen Stamm- / Stroma-Zellen aus humanen Placenta Gewebe gewonnen

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Isolieren von Mesangiogenic Progenitorzellen (MPL) aus menschlichem Knochenmark

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Zeitliche Reihenfolge der dynamischen Ausdruck von Daten aus Detaillierte Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolierung und Charakterisierung von mesenchymalen Stromazellen aus Nabelschnur und Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

Vorbereitung von Plasmamembran-Vesikeln aus Knochenmark Mesenchymale Stammzellen für potentielle Zytoplasma-Ersatztherapie

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioengineering von humanisierten Knochenmark-Mikroumgebungen in der Maus und ihre Visualisierung durch Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolation, Kultur und Differenzierung von Stromazellen Knochenmarkzellen und Osteoklasten-Vorläufer von Mäusen

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

Isolierung und Charakterisierung von mesenchymaler Stromazellen Herzzellen von Endomyocardial bioptischen Proben von Arrhythmogenic Kardiomyopathie Patienten

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...