Name: isolation of endothelial progenitor cells from human umbilical cord blood
Uploaded: 2017-09-14T14:00:00.000+00:00
Duration: 7 min 26 s
Description: Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Dieses Material basiert auf Arbeit, unterstützt von der National Science Foundation Grant No. CMMI-1452943 und von der University of Arkansas Ehrenhochschule. Wir möchten auch die Arkansas Nabelschnurblutbank für die uns mit Schnur Blut Einheiten zu erkennen.

dieser Forschung wurde mit Zustimmung der University of Arkansas Institutional Review Board (Zulassungsnummer 16-04-722) durchgeführt. Nabelschnur Blut Einheiten wurden in Citrat Phosphat Traubenzucker (CPD) Lösung in Arkansas Nabelschnurblutbank und Einheiten, die nicht die Voraussetzung für die Lagerung für die Forschung gespendet wurden. Cord Blood Einheiten waren couriered im Labor innerhalb von 24 h Kollektion bei Umgebungstemperaturen.
 1. Isolation der endothelialen Vorläuferzellen aus Nabelschnurblut <ol><li>Vorbereitung der Reagenzien.
	<ol><li>Ergänzt bereiten EGM durch Zugabe von endothelialen basal Medium (EBM), 10 % fetalen bovine Serum (FBS) mit 20 ng/mL des Insulin-Like Growth Factor (IGF), 1 µg/mL Ascorbinsäure, 5 ng/mL rekombinanten menschlichen epidermalen Wachstumsfaktor (RhEGF), 22,5 µg/mL Heparin, und 0,5 ng/mL vaskulären endothelialen Wachstumsfaktor (VEGF), zusammen mit 10 ng/mL von rekombinanten menschlichen Fibroblasten-Wachstumsfaktor B (RhFGF-B), 0,2 µg/mL Hydrocortison und 2 % Penicillin-Streptomycin-Glutamin. Sterilisieren Sie das Kulturmedium mit einem 0,2 µm Polyethersulfone (PES) Membrane Vakuumfilter.</li>
		<li>Vorbereiten Ratte tail ich Kollagen-Lösung mit einer Konzentration von 50 µg/mL mit 0,02 N Essigsäure. Diese Lösung mit einem 0,2 µm Spritze Filter sterilisieren.</li>
		<li>Pre-Mantel-6-Well-Platten mit 2 mL der vorbereiteten Ratte tail ich Kollagen-Lösung für jedes gut. Legen Sie sie in einem Inkubator auf 5 % CO 2 und 37 ° C für mindestens 1 h vor der Aussaat gehalten.</li>
		<li>Verdünnen etwa 25 mL Nabelschnurblut mit Hanks ausgewogen Salz Lösung (HBSS) in einer Konzentration von 1:1.</li>
		<li>Vorbereiten-Radio-Immuno-Niederschlag assay (RIPA) Puffer mit 150 mM Natrium-Chlorid, 1 % Triton x-100, 0,5 % Natrium Deoxycholate, 1 mM β-Glycerophosphat, 0,1 % SDS und 50 mM Tris (pH 8,0).</li>
	</ol></li> <li>Isolation von endothelialen Progenitorzellen.
	<ol><li>Warm All vorbereitet Reagenzien in einem Wasserbad auf 37 ° C vor der Isolierung gehalten.</li>
		<li>20 mL Dichte Gradienten mittlere Reagenz hinzufügen eine 50 mL Zentrifugenröhrchen.</li>
		<li>Sorgfältig Schicht 20 mL verdünnten Nabelschnurblut in der Dichte-Gradienten-Medium ohne diese Schicht mit dem Ziel, der Pipette auf den Seitenwänden des Rohres mit Zentrifugenröhrchen.</li>
		<li>Einzelteilen anhand ihrer Dichte ( Abbildung 1) Blut durch Zentrifugieren bei 800 X g für 30 min bei Raumtemperatur mit den Bremsen des Rotors Zentrifuge ab. Die verschiedenen Schichten nicht stören.</li>
		<li>Entfernen der Plasmaschicht durch pipettieren sie sorgfältig aus der Tube ohne zu stören andere Zelle, die Schichten bis die Pipettenspitze erreichen das MNC ist überlagern (siehe Abbildung 1). Entsorgen Sie das entfernte Plasma. 
		Hinweis: Bei der Verwendung von Pipettenspitzen, um das Plasma zu entfernen, lassen Sie eine kleine Schicht von Plasma über den MNC-Schicht ( Abbildung 1), um Störungen zu reduzieren.</li>
		<li>Sammeln die buffy Mantel, den die multinationalen Konzerne mit einer Spritze ausgestattet, ein 18-Gauge-Nadel enthält und überträgt es auf eine frische Zentrifugenröhrchen.</li>
		<li>Hinzufügen einem gleichen Volumen der EBM die gesammelten MNCs. Zentrifuge diese Rohre bei 500 X g für 10 min bei 4 ° C. verwerfen des Überstands. 
		Hinweis: Die Zelle-Pellet erscheinen rot wegen der Anwesenheit von roten Blutkörperchen/Erythrozyten.</li>
		<li>5 mL Ammoniumchlorid-Lösung für die Erythrozyten lysiert hinzugeben. Dieses Rohr für ca. 5-10 min, mit gelegentlichen schütteln auf Eis brüten. 
		Hinweis: Vorsicht getroffen werden während dieses Schrittes nicht überschreiten die Zeitdauer, wie es für die multinationalen Konzerne schädlich sein kann.</li>
		<li>Schläuche am 500 X g für 10 min bei 4 ° C zentrifugiert und den Überstand entsorgen. Wenn Erythrozyten bestehen bleiben, wiederholen Sie die Schritte 1.2.8 und 1.2.9, bis keine rote Farbe in das Pellet beobachtet wird.</li>
		<li>Das Pellet in vorbereiteten EGM Aufschwemmen und verwenden die Hemocytometer, die multinationalen Konzerne zählen.</li>
		<li>Aspirat Ratte tail ich Kollagen beschichtet 6-Well-Platte (aus Schritt 1.1.3) und waschen Sie die Brunnen dreimal mit 1 x Phosphat-gepufferte Kochsalzlösung (PBS).</li>
		<li>Samen das MNC-Pellet Nukleinsäuretablette in EGM auf die Kollagen-Platte in einer Konzentration von 1 x 10 7 MNCs in jede Vertiefung. Legen Sie sie in das 37 ° C, 5 % CO 2-Inkubator.</li>
		<li>Nach 24 h, aspirieren Sie das Medium und die Brunnen einmal mit EGM zu waschen. Jede Vertiefung 3 mL EGM Medium hinzu und legen Sie sie zurück in das 37 ° C, 5 % CO 2-Inkubator.</li>
		<li>Ergänzen die Platte mit frischen EGM Medium täglich für 7 Tage. Nach 7 Tagen das EGM-Medium verändern jeden zweiten Tag.</li>
		<li>Mit einem Hellfeld Mikroskop Bilder von der 6-Well-Platte nehmen jeden Tag, um das Fortschreiten der Endothelzellen Kolonien zu verfolgen. Markieren Sie die Platte, wo die Kolonien entstehen, deren Wachstum nachzuverfolgen. 
		Hinweis: Es dauert in der Regel zwischen 5 und 9 Tage für die Kolonien in der Kultur angezeigt werden.</li>
	</ol></li> <li>Ausbau der endothelialen Vorläuferzellen.
	<ol><li>Mantel der t-25-Kultur-Küvette mit Ratte tail ich Kollagen (50 µg/mL) und legen Sie sie in das 37 ° C, 5 % CO 2 Inkubator für mindestens 60 min. Absaugen und waschen die t-25 Zelle Kultur Küvette dreimal mit 1 X PBS vor der Aussaat Zellen.</li>
		<li>Trypsinize die Endothelzellen Kolonien, wenn sie etwa 3 mm groß mit 150 µL 0,05 % Trypsin-EDTA-Lösung in jede Vertiefung zu erreichen. Setzen Sie die 6-Well-Platte wieder in 37 ° C, 5 % CO 2 Inkubator für 2-3 min.</li>
		<li>Klopfen die 6-Well-Platte um die angeschlossenen Zellen verdrängen. Sofort 2 mL der EGM und Wiederherstellen der Zellen in einem Zentrifugenröhrchen 15 mL.</li>
		<li>Der Zellen unter Verwendung einer Hemocytometer zählen und rechnen die ursprünglichen Aussaatdichte.</li>
		<li>Spin der Zentrifuge 15 mL Röhrchen bei 400 X g für 5 min. Aufschwemmen der Zelle Pellet in EGM und Samen den t-25 Kolben mit ~ 500.000 Zellen. Markieren Sie dieses Fläschchen als Durchgang 1 und t-25 Kolben wieder in 37 ° C, 5 % CO 2-Inkubator. 
		Hinweis: Zellen können in EBM und DMEM, das Wachstum mit EGM vergleichen vernickelt werden.</li>
		<li>Für weitere Passagen, wenn der Kolben Zusammenfluss von 90 % erreicht, führen Sie Schritte 1.3.1 - 1.3.5 und reseed Zellen 10 4 Zellen/cm 2 T-75 oder T-175 Zellkulturflaschen. Fahren Sie mit der Charakterisierung der isolierten Zellen (Schritte 2 und 3).</li>
	</ol></li></ol> 2. Charakterisierung der isolierten Zellen mittels Western Blotting <ol><li>Lyse hinzufügen 50-100 µL Puffer bei jedem Durchgang Handlungsschritte auf Ausbau der isolierten Zellen zu charakterisieren. Proteine von etwa 500.000 oder mehr Zellen sammeln.</li>
	<li>Pipette oben und unten kräftig zu platzen die Zellen und Proteine. Sammeln und übertragen den Puffer zu einem Microcentrifuge Schlauch.</li>
	<li>Spin Microcentrifuge Schlauch bei 500 X g und den Überstand sorgfältig auf einen neuen Microcentrifuge Schlauch übertragen. Benennen und Speichern der Rohre bei-80 ° C für die Langzeitspeicherung.</li>
	<li>Quantifizieren die Menge der Proteine in jedem Röhrchen verwenden entweder Bradford &#39; s oder BCA assay 27 , 28 , 29.</li>
	<li>Führen durch Western Blot mit Standardverfahren 27 , 28 , 29. 5 µg Gesamt-Protein für den Ausdruck von CD31, CD34, VEGFR2 und α-SMA zu analysieren. Α-Tubulin für die Normalisierung der Daten verwenden.</li>
</ol> 3. Indirekte Immunfluoreszenz <ol><li>18 mm Glasdeckgläser in 50 % igem Ethanol beschallen und trocknen Sie sie. Verlassen sauberen Deckgläsern in ein 12-Well-Platte über Nacht in der Biosicherheits-Haube mit einem UV-Licht auf um sie zu sterilisieren.</li>
	<li>Mantel Deckgläsern mit 50 & #181; g/mL Ratte tail ich Kollagen und übertragen Sie die Platte auf einem 37 ° C, 5 % CO 2 Inkubator für mindestens 60 min.</li>
	<li>Aspirieren das Kollagen und die 12-Well-Platte, die Deckgläsern waschen 1 mL 1 X PBS hinzufügen. 1 X PBS Aspirieren und zweimal wiederholen.</li>
	<li>Samen 250.000 kultiviert EPC in jedem gut und sie zurück in das 37 ° C, 5 % CO 2 Inkubator für mindestens 3 Tage vor Durchführung Immunostaining.</li>
	<li>Waschen die Platte 3 Mal mit 1 X PBS. Fügen Sie 1 mL eiskaltes Methanol in jede Vertiefung und Inkubation bei Raumtemperatur 15 Minuten, die Zellen zu beheben.</li>
	<li>Führen Immunostaining mit einem Standardprotokoll 27 , 28 , 29. Die Antikörper bei ihren entsprechenden Verdünnungen verwenden (z. B. CD31 (01:20), CD34 (01:50), α-SMA (1: 100) und VEGFR2 (01:50)).</li>
	<li>Bilder von den Immunostained Deckgläsern mit einem Epifluoreszenz Mikroskop nehmen.</li>
</ol>

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Wie bereits erwähnt, anhaftende EPCs besitzen eine Kopfsteinpflaster-Morphologie. Unsere isolierten MNCs entwickelte sich von einer spindelförmigen Zelle Kolonie (Abbildung 2A-2D) in den frühen Stadien zu einer gepflasterten Kolonie (Abbildung 2E-2F) über einen Zeitraum von zehn Tagen in Kultur. EPC haben unterschiedlich von verschiedenen Forschungsgruppen, nämlich als späten endothelial Progenitor Zellen...

Mehrere Forscher untersuchten die Eigenschaften und das Potential der menschlichen EPCs5,10,11,12,13. EPC können beschrieben werden als Zellen, die Fähigkeit zur Einhaltung endothelial Gewebe an Standorten von Hypoxie, Ischämie, Verletzungen oder Tumorbildung und tragen zur Bildung von neuen vaskulären Strukturen4,14in Umlauf. Ihre beobachteten Beteiligung an Neovaskularisation, in Form von postnatale Vaskulogenese, führte zu einem Verständnis der Pathophysiologie der diese Zellen und deren Anwendung in therapeutischen Anwendungen4,15, 16. die Anzahl der EPC im Einzelfall nachweislich korreliert mit Herz-Kreislauf-Pathologie9,15,16,17,18,19 ,20. Andere Studien haben auch differenzierte EPC in einem Ventil Fibroblasten-ähnlichen Phänotyp und schlug vor, dass diese Zellen für Gewebe-Engineering Herz Ventile7,21verwendet werden könnten.
Bestimmte Zelle Oberfläche Moleküle EPCs isolieren musste wurden aufgrund von Unstimmigkeiten zwischen den Untersuchungen4nicht eindeutig identifiziert. Die Haftung der multinationalen Konzerne zu einer bestimmten Matrix, mit der Exposition gegenüber einer Vielzahl von Kulturbedingungen durchgeführt worden durch mehrere Gruppen1,17,22,23, was darauf hindeutet, dass vermeintliche EPCs kann unterschiedlichen phänotypische Eigenschaften anzeigen. Zu diesen Eigenschaften gehören einen Mangel an phagocytotic Fähigkeit, Rohr-Bildung in Matrigel und die Aufnahme von Low-Density-Lipoproteine Dil acetyliert. Die hohe klonogenen und proliferative Potential sind zwei Eigenschaften, die mit dem EPC hierarchisierte5werden können. EPC können auch in-vitro- Tubuli wenn mit menschlichen fetalen Lunge Fibroblasten4cocultured bilden. Diese Zellen sind Endothelzellen Oberflächenmarker zum Ausdruck zu bringen und einige der hämatopoetischen Marker13,24,25Teilen bekannt. Die positiv ausgedrückt Marker, die weithin für Phänotypisierung EPCs akzeptiert werden sind CD31, CD34, vaskulären endothelialen Wachstumsfaktor-Rezeptor 2 (VEGFR2), von-Willebrand-Faktor (vWF), CD133, c-Kit und vaskulären endothelialen Cadherin (VE-Cadherin)4 , 18. Zellen, die CD90, CD45, CD14, CD115 oder Alpha-Glattmuskel Aktin (α-SMA) Co ausdrücken gelten nicht als EPC wegen ihrer begrenzten proliferative potenzielle, Fähigkeit, Phagozytose von Bakterien und die Unfähigkeit, de Novo menschlichen bilden Schiffe in Vivo4,7. Dieser Artikel beschreibt ein modifiziertes Protokoll für die Isolierung von endothelialen Vorläuferzellen aus menschlichen Nabelschnurblut ohne die Notwendigkeit für jede Zelle sortieren Protokolle. In diesem Artikel verwendet CD31, CD34 und VEGFR2 als die positive Markierungen mit α-SMA als negativer Indikator.
In diesem Artikel schlagen wir vor, dass eine Methode zur Isolierung und Kultivierung von endothelialen Vorläuferzellen aus Nabelschnurblut ohne Zelle sortieren mit endothelialen Wachstumsmedium ergänzt mit Wachstumsfaktoren (EGM) spezialisiert. Diese EGM vaskulären endothelialen Wachstumsfaktor (VEGF) und enthält Fibroblasten-Wachstumsfaktor (FGF), die das Überleben, Proliferation und Migration von Endothelzellen26zu verbessern. Es enthält auch Ascorbinsäure, die für die Aufrechterhaltung der Kopfsteinpflaster-Morphologie der Zellen verantwortlich ist; Insulin-ähnliche Wachstumsfaktor-1 (IGF-1), die Angiogenese und wandernden Funktion bietet; und Heparin, wodurch verbesserte Langzeitstabilität von Wachstumsfaktoren in der mittleren26. Andere Wachstumsfaktoren hinzugefügt, um die endotheliale Zellkulturmedium umfasst die Supplementation mit epidermalen Wachstumsfaktor (EGF), die hilft, anregende Zell-Proliferation und Differenzierung und Hydrocortison, die die Zellen EGF26 sensibilisiert . Wir zeigen, dass die Nutzung dieses Mediums spezifischen Wachstumsraten höhere Anzahl von EPC im Vergleich zu Endothelzellen basal Medium (EBM) oder Dulbeccos geändert Eagle Medium (DMEM ergibt).

<table><tbody><tr><td>&lt;strong&gt;A) For isolation and culturing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Pencillin-Streptomycin-Glutamine (100x)</td><td>Thermofisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Heatlhcare</td><td>17-1440-02</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermofisher Scientific</td><td>14170-112</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Stem Cell Technologies</td><td>7850</td><td></td></tr><tr><td>1x Phosphate Buffer Saline</td><td>Thermofisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Rat Tail I Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Amresco</td><td>0714-500ML</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermofisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Thermofisher Scientific</td><td>10566-016</td><td></td></tr><tr><td>&lt;strong&gt;B) Antibodies and cell lysates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31&amp;nbsp;</td><td>Abcam</td><td>ab28364</td><td>1:250 dilution&amp;nbsp; for Western blotting</td></tr><tr><td>CD34</td><td>Santa Cruz Biotechnology</td><td>sc-7045</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-SMA</td><td>abcam</td><td>ab5694</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-tubulin</td><td>abcam</td><td>ab7291</td><td>1:2,500 dilution for Western blotting</td></tr><tr><td>VEGFR2</td><td>abcam</td><td>sc504</td><td>1:100 dilution for Western blotting</td></tr><tr><td>Human umbilical vein endothelial cell lysate</td><td>Santa Cruz Biotechnology</td><td>sc24709&amp;nbsp;</td><td></td></tr><tr><td>Valve interstitial cell lysate</td><td></td><td></td><td>Primary cell line cultured from own lab and lysed with RIPA buffer</td></tr><tr><td>&lt;strong&gt;C) Western blotting and immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Tris/Glycine/SDS buffer</td><td>Biorad</td><td>161-0772</td><td>Used as running buffer</td></tr><tr><td>10x Tris/Glycine buffer</td><td>Biorad</td><td>161-0771</td><td>Used as transfer buffer</td></tr><tr><td>Immobilon-FL transfer membrane</td><td>Merck Millipore</td><td>IPFL0010</td><td>This is a PVDF transfer membrane that has 45 &amp;micro;m pore size and is mentioned in the protocol as western blot membrane</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>161-0747</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>161-0710</td><td></td></tr><tr><td>10% Criterion TGX precast gel</td><td>Biorad</td><td>5671033</td><td></td></tr><tr><td>Prolong Gold antifade</td><td>Thermofisher Scientific</td><td>P36930</td><td>Used for mounting immunostained coverslips for long term storage</td></tr><tr><td>Methanol</td><td>VWR Analytical</td><td>BDH1135-4LP</td><td></td></tr></tbody></table>

isolation of endothelial progenitor cells from human umbilical cord blood

Die Existenz von endothelialen Progenitorzellen (EPC) im peripheren Blut und seine Einbindung in Vaskulogenese wurde zuerst von Ashara und Kollegen1berichtet. Später, dokumentiert andere die Existenz von ähnlichen Arten von EPC aus dem Knochenmark2,3. Vor kurzem hatte Yoder und Ingram zeigte, dass Nabelschnurblut EPCs abgeleitet ein höheres proliferative Potential im Vergleich zu denen von Erwachsenen peripheren isoliert Blut-4,5,6. Neben der postnatalen Vaskulogenese beteiligt zu sein, haben EPC auch Versprechen als eine Zelle Quelle für die Erstellung von Tissue-Engineering Gefäß- und Herz-Ventil Konstrukte7,8gezeigt. Verschiedenen Isolierung Protokolle sind vorhanden, von denen einige beinhalten Zellsortierung mononukleären Zellen (MNU) abgeleitet aus den Quellen erwähnt mit Hilfe von Endothelzellen und hämatopoetischen Marker oder Kultivierung dieser multinationalen Unternehmen mit spezialisierten endothelial Wachstum Medium, oder eine Kombination dieser Techniken-9. Hier präsentieren wir Ihnen ein Protokoll für die Isolierung und Kultur des EPC mit spezialisierten Endothelzellen Medium ergänzt mit Wachstumsfaktoren, ohne den Einsatz von Immunosorting, gefolgt von der Charakterisierung von der isolierten Zellen mittels Western blotting und Immunostaining.

Isolation und Ausbau der endothelialen Vorläuferzellen: Ein Schaltplan (Abbildung 1) steht zur Verfügung, zeigt das gesamte Protokoll. Die verschiedenen Blut Komponentenschichten wurden nach Dichte Gradienten Zentrifugieren der menschlichen Nabelschnurblut mit Dichte Gradienten Medium beobachtet. Bei der Aussaat MNCs auf die Kollagen-behandelten Platten, wurde erstmals Auswuchs von Kolonien zwischen 5 Tagen und 7 (Ab...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Das Ziel dieses Protokolls ist, endotheliale Vorläuferzellen aus Nabelschnurblut zu isolieren. Einige der Anwendungen sind mit diesen Zellen als Biomarker für die Identifizierung von Patienten mit Herz-Kreislauf-Risiko, Behandlung von ischämischen Erkrankungen und Erstellen von Tissue-Engineering Gefäß- und Herz-Ventil konstruiert.

Isolierung der endothelialen Vorläuferzellen aus menschlichen Nabelschnurblut

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

isolation-endothelial-progenitor-cells-from-human-umbilical-cord

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.1K Aufrufe.  University of Arkansas. Das Ziel dieses Protokolls ist, endotheliale Vorläuferzellen aus Nabelschnurblut zu isolieren. Einige der Anwendungen sind mit diesen Zellen als Biomarker für die Identifizierung von Patienten mit Herz-Kreislauf-Risiko, Behandlung von ischämischen Erkrankungen und Erstellen von Tissue-Engineering Gefäß- und Herz-Ventil konstruiert.

Serie: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Direkte Induktion von humanen neuralen Stammzellen aus peripherem Blut hämatopoetischen Vorläuferzellen

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Forschung

Entwicklungsbiologie

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolierung humaner Nabelschnur-Endothelzellen (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biologie

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolierung von murinen embryonalen Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Isolierung von humanen Nabelschnur-Endothelzellen und ihre Verwendung in der Studie von neutrophilen Transmigration unter Strömungsbedingungen

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Immunologie und Infektion

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolierung von Precursor B-Zell-Subsets aus Nabelschnurblut

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Panmyeloische Differenzierung von humanem Cordblood abgeleitet CD34+ hämatopoetische Stamm- und Vorläuferzellen

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Isolierung von endothelialen Vorläuferzellen von gesunden Freiwilligen und ihre Migratory Potential Beeinflusst von Serumproben nach einer Herzoperation

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Medizin

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Phänotypische und funktionelle Charakterisierung von Endothelial Kolonie-bildenden Zellen aus humanem Nabelschnurblut

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Ex-Vivo Expansion von hämatopoetischen Stammzellen aus menschlichen Nabelschnur Blut abgeleitet CD34+ Zellen mit Valproinsäure

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Isolierung der endothelialen Vorläuferzellen aus menschlichen Nabelschnurblut

Zusammenfassung

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