Questo materiale si basa su lavori sostenuta dalla National Science Foundation Grant No. CMMI-1452943 e dall'Università di Honors di Università dell'Arkansas. Vorremmo anche riconoscere l'Arkansas Cord Blood Bank per averci fornito unità cordonali.

questa ricerca è stata condotta con l'approvazione dell'Università di Arkansas Institutional Review Board (numero di omologazione 16-04-722). Unità di sangue del cordone ombelicale sono stati raccolti in soluzione di citrato fosfato destrosio (CPD) presso l'Arkansas Cord Blood Bank, e unità che non soddisfano il requisito per l'archiviazione sono stati donati per la ricerca. Unità cordonali erano corriere al laboratorio entro 24 h dalla raccolta a temperatura ambiente.
 1. isolamento di cellule endoteliali progenitrici dal sangue del cordone ombelicale <ol><li>preparazione dei reagenti.
	<ol><li>EGM preparare aggiungendo sostanza basale endoteliale (EBM) al 10% siero bovino fetale (FBS) completati con 20 ng/mL del fattore di crescita insulino-simile (IGF), 1 µ g/mL di acido ascorbico, fattore di crescita ricombinante umano epidermico 5 ng/mL (rhEGF), 22,5 µ g/mL eparina e 0,5 ng/mL endoteliale fattore di crescita vascolare (VEGF), insieme a 10 ng/mL di fattore di crescita del fibroblasto umano ricombinante B (rhFGF-B), idrocortisone di 0,2 µ g/mL e 2% penicillina-streptomicina-glutammina. Sterilizzare il terreno di coltura utilizzando un filtro vuoto a membrana in polietersulfone (PES) 0,2 µm.</li>
		Ratto <li>preparare io coda soluzione di collagene ad una concentrazione di 50 µ g/mL utilizzando 0,02 N di acido acetico. Sterilizzare questa soluzione utilizzando un filtro per siringa 0,2 µm.</li>
		<li>Pre-cappotto 6-pozzetti con 2 mL di preparato ratto ho coda soluzione di collagene per ciascun pozzetto. Metterli in un incubatore mantenuto a 5% CO 2 e 37 ° C per almeno 1 h prima della semina.</li>
		<li>Diluire circa 25 mL di sangue del cordone ombelicale con Hanks bilanciato sale soluzione (HBSS) ad una concentrazione di 1:1.</li>
		<li>Preparare radio-immuno precipitazione (RIPA) tampone utilizzando 150 mM di cloruro di sodio, 1% Triton X-100, 0,5% sodio desossicolato, β-glicerofosfato di 1 mM, 0,1% SDS e 50 mM Tris (pH 8.0) per il test.</li>
	</ol></li> <li>Isolamento di cellule progenitrici endoteliali.
	<ol><li>Caldo tutti preparati i reagenti in un bagno d'acqua mantenuto a 37 ° C prima dell'isolamento.</li>
		<li>Aggiungere 20 mL di reagente medio gradiente di densità in una provetta da centrifuga 50ml.</li>
		<li>Attentamente strato 20 mL di sangue cordonale diluito la provetta da centrifuga contenente medio gradiente di densità senza rompere questo strato puntando la pipetta verso le pareti laterali del tubo.</li>
		<li>Separare i componenti del sangue basata sulla loro densità ( Figura 1) mediante centrifugazione a 800 x g per 30 min a temperatura ambiente, con i freni del rotore della centrifuga fuori. Non disturbare i diversi strati.</li>
		<li>Rimuovere lo strato di plasma pipettando attentamente fuori dal tubo senza disturbare altri cell strati fino a quando la punta della pipetta è in grado di raggiungere il MNC strato (Vedi Figura 1). Scartare il plasma rimosso. 
		Nota: Durante l'utilizzo di puntali per rimuovere il plasma, lasciare un piccolo strato di plasma sopra lo strato MNC ( Figura 1) per ridurre i disturbi.</li>
		<li>Raccogliere il buffy-coat contenente i PTM usando una siringa graduata per un ago calibro 18 e trasferirlo in una provetta da centrifuga freschi.</li>
		<li>Aggiungere un volume uguale di EBM alla centrifuga MNCs. raccolti questi tubi a 500 x g per 10 min a 4 ° C. scartare il sopranatante. 
		Nota: Il pellet cellulare apparirà rosso a causa della presenza di globuli rossi/eritrociti.</li>
		<li>Aggiungere 5 mL di soluzione di cloruro di ammonio per lisare le cellule di anima rosse. Incubare la provetta in ghiaccio per 5-10 minuti, con agitazione occasionale. 
		Nota: Prestare attenzione durante questo passaggio di non superare la durata di tempo, come può essere dannoso per i PTM.</li>
		<li>Centrifugare le provette a 500 x g per 10 min a 4 ° C e scartare il surnatante. Se persistono gli eritrociti, ripetere i passaggi 1.2.8 e 1.2.9 Nessun colore rosso è osservato nel pellet.</li>
		<li>Risospendere il pellet in EGM preparati e utilizzare l'emocitometro per contare i PTM.</li>
		<li>Aspirato il ratto di coda ho piastra a 6 pozzetti rivestite con collagene (dal punto 1.1.3) e lavare i pozzetti tre volte con 1x tampone fosfato salino (PBS).</li>
		<li>Del seme il pellet MNC risospeso in EGM alla piastra di collagene ad una concentrazione di 1 x 10 7 MNCs in ciascun pozzetto. Posizionarlo a 37 ° C, 5% CO 2 incubator.</li>
		<li>Dopo 24 h, il mezzo di aspirare e lavare i pozzetti una volta con EGM. Aggiungere 3 mL di terreno EGM in ciascun pozzetto e posizionarlo torna a 37 ° C, 5% CO 2 incubator.</li>
		<li>Riempire la piastra con mezzo EGM fresco ogni giorno per 7 giorni. Dopo 7 giorni, cambiare il mezzo EGM alterni.</li>
		<li>Usando un microscopio a campo chiaro, prendere le immagini della piastra 6 pozzetti ogni giorno per monitorare la progressione delle colonie delle cellule endoteliali. Contrassegnare la piastra dove le colonie derivano per tenere traccia della loro crescita. 
		Nota: Prende solitamente fra 5 e 9 giorni per le colonie a comparire nella cultura.</li>
	</ol></li> <li>Espansione di cellule progenitrici endoteliali.
	<ol><li>Cappotto il matraccio di cultura T-25 con ratto coda ho collagene (50 µ g/mL) e posizionarlo a 37 ° C, 5% CO 2 incubatore per almeno 60 min. aspirare e lavare il matraccio di cultura cellulare T-25 tre volte con PBS 1x prima della semina delle cellule.</li>
		<li>Tripsinizzano le colonie delle cellule endoteliali quando raggiungono circa 3 millimetri nel formato utilizzando 150 µ l di soluzione di tripsina-EDTA 0,05% in ciascun pozzetto. Inserire nuovamente la piastra a 6 pozzetti i 37 ° C, 5% CO 2 incubatrice per un minimo di 2-3</li>
		<li>Toccare delicatamente la piastra 6 pozzetti per sloggiare le cellule allegate. Immediatamente aggiungere 2 mL di EGM e ripristinare le cellule in una provetta da centrifuga da 15 mL.</li>
		<li>Contare le celle utilizzando un emocitometro e calcolare la densità di semina iniziale.</li>
		Tubi di <li>spin la centrifuga 15 mL a 400 x g per 5 min. Risospendere il pellet cellulare in EGM e seminare la bottiglia di T-25 con ~ 500.000 cellule. Contrassegnare questa boccetta come passaggio 1 e posizionare il pallone T-25 torna a 37 ° C, 5% CO 2 incubator. 
		Nota: Le cellule possono essere placcate in EBM e DMEM per confrontare la crescita con EGM.</li>
		<li>Per ulteriori passaggi, quando il pallone raggiunge 90% confluenza, attenersi alla seguente procedura 1.3.1 - 1.3.5 e reinizializzare le cellule 10 4 cellule/cm 2 su T-75 o T-175 matracci di cultura cellulare. Procedere alla caratterizzazione di cellule isolate (passaggi 2 e 3).</li>
	</ol></li></ol> 2. Caratterizzazione di isolato cellule mediante Western Blotting <ol><li>Aggiungi 50-100 µ l di lysis buffer ad ogni passaggio quando seguendo la procedura di espansione per caratterizzare le cellule isolate. Raccogliere le proteine da circa 500.000 o più cellule.</li>
	<li>Pipettare vigorosamente su e giù a rottura delle cellule e rilasciare le proteine. Raccogliere e il buffer di trasferimento ad un tubo del microcentrifuge.</li>
	<li>Spin il tubo del microcentrifuge a 500 x g e attentamente trasferire il surnatante ad un nuovo tubo del microcentrifuge. Etichetta e memorizzare le provette a-80 ° C per la conservazione a lungo termine.</li>
	<li>Quantificare la quantità di proteine in ogni provetta utilizzando entrambi Bradford &#39; s o BCA dosaggio 27 , 28 , 29.</li>
	<li>Eseguire Western blotting usando le procedure standard 27 , 28 , 29. Analizzare 5 µ g di proteina totale per l'espressione di CD31, CD34, VEGFR2 e α-SMA. Utilizzare α-tubulina per normalizzare i dati.</li>
</ol> 3. Immunofluorescenza indiretta <ol><li>sottoporre ad ultrasuoni-18mm vetrini coprioggetti in etanolo al 50% e asciugarli. Lasciare i coprioggetti puliti in un piatto di 12-pozzetti pernottamento nella cappa di sicurezza biologica con un UV luce su per sterilizzarli.</li>
	<li>Cappotto lamelle con 50 & n. 181; g/mL di ratto coda ho collagene e trasferire la piastra a 37 ° C, 5% CO 2 incubatore per almeno 60 min.</li>
	<li>Aspirare il collagene e aggiungere 1 mL di PBS 1x alla piastra 12-pozzetti per lavare i vetrini coprioggetti. Aspirare il PBS 1X e ripetere due volte.</li>
	<li>Seme 250.000 coltivate EPCs in ciascun pozzetto e il posto nuovamente a 37 ° C, 5% CO 2 incubatore per almeno 3 giorni prima di effettuare immunostaining.</li>
	<li>Lavare la piastra 3 volte con PBS 1X. Aggiungere 1 mL di metanolo ghiacciato in ogni pozzetto e incubare a temperatura ambiente per 15 minuti per fissare le cellule.</li>
	<li>Eseguire immunostaining usando un protocollo standard 27 , 28 , 29. Utilizzare gli anticorpi alle loro appropriate diluizioni (ad es., CD31 (01:20), CD34 (01:50), α-SMA (1: 100) e VEGFR2 (01:50)).</li>
	<li>Prendere le immagini di lamelle immunostained utilizzando un microscopio a epifluorescenza.</li>
</ol>

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Come accennato in precedenza, aderente EPCs possiedono una morfologia di ciottoli. Nostro MNCs isolato progredito da una colonia di cellule fusiformi (Figura 2A-2D) nelle prime fasi di una colonia di ciottoli (Figura 2E-2F) per un periodo di dieci giorni nella cultura. EPC sono state etichettate in modo diverso dai gruppi di ricerca differenti, vale a dire come ritardati di cellule progenitrici endoteliali10</s...

Parecchi ricercatori hanno studiato le caratteristiche e le potenzialità di umano EPCs5,10,11,12,13. EPC possono essere descritto come fare circolare le cellule che hanno la capacità di aderire al tessuto endoteliale nei siti di ipossia, ischemia, lesioni o di formazione del tumore e contribuiscono alla formazione di nuove strutture vascolari4,14. Loro coinvolgimento osservato nella neovascolarizzazione, sotto forma di vasculogenesi postnatale, ha condotto ad una comprensione della patofisiologia di queste cellule e il loro uso in applicazioni terapeutiche4,15, 16. il numero di EPCs in un individuo è stato indicato per essere correlati con patologia cardiovascolare9,15,16,17,18,19 ,20. Altri studi hanno anche differenziato EPCs in un fenotipo fibroblasto-come valvola e proposto che queste cellule potrebbero essere utilizzate per l'ingegneria dei tessuti cuore valvole7,21.
Le molecole di superficie determinata cella necessari per isolare EPCs non sono stati chiaramente identificate a causa di discrepanze tra le indagini4. L'adesione delle MNCs per una certa matrice, con l'esposizione a una varietà di condizioni di coltura, è stata eseguita da diversi gruppi1,17,22,23, suggerendo che presunto EPCs può visualizzare le proprietà fenotipiche differenti. Queste proprietà includono la mancanza di abilità phagocytotic, la formazione del tubo in Matrigel e l'assorbimento delle lipoproteine a bassa densità di Dil-acetilato. L'alta clonogenic e potenziale proliferativo sono due proprietà con cui EPCs può essere gerarchizzato5. EPCs possono anche formare in vitro i tubuli quando cocultured con fibroblasti di polmone fetale umano4. Queste cellule sono conosciute per esprimere gli indicatori di superficie delle cellule endoteliali e di condividere alcuni dei marcatori ematopoietici13,24,25. I marcatori espressi positivamente che sono ampiamente accettati per la fenotipizzazione EPCs sono CD31, CD34, fattore di crescita endoteliale vascolare receptor 2 (VEGFR2), von Willebrand Factor (vWF), CD133, c-Kit e vascolare endoteliale caderina (VE-caderina)4 , 18. non sono considerate essere EPCs a causa della loro limitata capacità proliferativa potenziali, di fagocitare batteri e incapacità di formare de novo umano cellule che co-esprimono CD90, CD45, CD14, CD115 o l'actina alfa-liscia del muscolo (α-SMA) vasi in vivo4,7. Questo articolo descrive un protocollo modificato per l'isolamento di cellule progenitrici endoteliali dal sangue del cordone ombelicale umano senza la necessità di qualsiasi cella ordinamento protocolli. In questo articolo abbiamo usato CD31, CD34 e VEGFR2 come gli indicatori positivi, con α-SMA come indicatore negativo.
In questo articolo, vi proponiamo un metodo di isolamento e coltura delle cellule progenitrici endoteliali da sangue del cordone ombelicale senza cella ordinamento utilizzando specializzato supplementato crescita endoteliale con fattori di crescita (EGM). Questo EGM contiene fattore di crescita endoteliale vascolare (VEGF) e fattore di crescita del fibroblasto (FGF), che migliorano la sopravvivenza, la proliferazione e la migrazione di cellule endoteliali26. Include anche l'acido ascorbico, che è responsabile del mantenimento della morfologia di ciottoli delle cellule; 1 fattore di crescita insulino-simile (IGF-1), che fornisce angiogenici e funzione migratori; e l'eparina, che provoca una migliore stabilità a lungo termine dei fattori di crescita nel medio26. Altri fattori di crescita aggiunti al terreno di coltura delle cellule endoteliali comprende il completamento con fattore di crescita epidermico (EGF), che aiuta a stimolare la proliferazione delle cellule e differenziazione e idrocortisone, che sensibilizza le cellule per EGF26 . Indichiamo che l'utilizzo di questo mezzo di crescita specifici rendimenti più alti numeri di EPCs rispetto al medio basale endoteliale (EBM) o per volta Eagle Medium (DMEM di Dulbecco).

<table width="1257">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="width:204px;height:20px;">A) For isolation and culturing</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:807px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">EGM-2 BulletKit</td>
			<td style="width:127px;">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 height="42">
			<td height="42" style="width:204px;height:42px;">Fetal Bovine Serum</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">26140079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Pencillin-Streptomycin-Glutamine (100X)</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">10378016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Ficoll-Paque</td>
			<td style="width:127px;">GE Heatlhcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Ammonium Chloride</td>
			<td style="width:127px;">Stem Cell Technologies</td>
			<td style="width:119px;">7850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">1X Phosphate Buffer Saline</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Rat Tail I Collagen</td>
			<td style="width:127px;">Corning</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Glacial Acetic Acid</td>
			<td style="width:127px;">Amresco</td>
			<td style="width:119px;">0714-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">HEPES buffer</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">10566-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:204px;height:20px;">B) Antibodies and cell lysates</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">CD31&#160;</td>
			<td style="width:127px;">Abcam</td>
			<td style="width:119px;">ab28364</td>
			<td>1:250 dilution&#160; for Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">CD34</td>
			<td style="width:127px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-7045</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">&#945;-SMA</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">ab5694</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">&#945;-tubulin</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">ab7291</td>
			<td>1:2500 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">VEGFR2</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">sc504</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Human umbilical vein endothelial cell lysate</td>
			<td style="width:127px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc24709&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Valve interstitial cell lysate</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:807px;">Primary cell line cultured from own lab and lysed with RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:204px;height:40px;">C) Western blotting and immunostaining</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10X Tris/Glycine/SDS buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0772</td>
			<td>Used as running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10X Tris/Glycine buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0771</td>
			<td>Used as transfer buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Immobilon-FL transfer membrane</td>
			<td style="width:127px;">Merck Millipore</td>
			<td style="width:119px;">IPFL0010</td>
			<td>This is a PVDF transfer membrane that has 45 &#181;m pore size and is mentioned in the protocol as western blot membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">4X Laemmli sample buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">2-mercaptoethanol</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10% Criterion TGX precast gel</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">5671033</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Prolong Gold antifade</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">P36930</td>
			<td>Used for mounting immunostained coverslips for long term storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Methanol</td>
			<td style="width:127px;">VWR Analytical</td>
			<td style="width:119px;">BDH1135-4LP</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of endothelial progenitor cells from human umbilical cord blood

L'esistenza di cellule progenitrici endoteliali (EPC) nel sangue periferico e il suo coinvolgimento nella vasculogenesi fu riportata da Ashara e colleghi1. Più tardi, altri documentata l'esistenza di tipi simili di EPCs provenienti dal midollo osseo2,3. Più recentemente, Yoder e Ingram ha mostrato che EPCs derivate da sangue del cordone ombelicale aveva un potenziale proliferativo più elevato rispetto a quelli isolati da adulto periferico sangue4,5,6. Oltre ad essere coinvolti nella vasculogenesi postnatale, EPCs inoltre hanno indicato la promessa come una fonte di cellule per la creazione di ingegneria tissutale vascolare e cuore valvola costrutti7,8. Esistono diversi protocolli di isolamento, alcuni dei quali coinvolgono l'ordinamento delle cellule delle cellule mononucleari (MNCs) derivate dalle fonti indicate in precedenza con l'aiuto di markers endoteliali ed ematopoietiche, o coltura questi MNCs con crescita endoteliale specializzata medio, o una combinazione di queste tecniche9. Qui, presentiamo un protocollo per l'isolamento e coltura di EPCs utilizzando specializzati endoteliale supplementato con fattori di crescita, senza l'uso di immunosorting, seguita dalla caratterizzazione di cellule isolate mediante Western blotting e immunostaining.

Isolamento ed espansione di cellule progenitrici endoteliali: Uno schema (Figura 1) viene fornito raffigurante il protocollo generale. I livelli di componenti del sangue diverso sono stati osservati dopo centrifugazione in gradiente di densità del sangue del cordone ombelicale con il mezzo di gradienti di densità. Al momento di semina MNCs sulle piastre collagene-trattati, la conseguenza delle colonie in primo luogo è stata osservata fr...

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

L'obiettivo del presente protocollo è quello di isolare le cellule progenitrici endoteliali da sangue del cordone ombelicale. Alcune delle applicazioni includono l'utilizzo di queste cellule come biomarcatore per identificare i pazienti con rischio cardiovascolare, trattamento delle malattie ischemiche, e costrutti di creazione valvola vascolare e cuore tessutale.

Isolamento di cellule endoteliali progenitrici dal sangue del cordone ombelicale umano

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

Research

JoVE Journal

Developmental Biology

11.0K Views.  University of Arkansas. L'obiettivo del presente protocollo è quello di isolare le cellule progenitrici endoteliali da sangue del cordone ombelicale. Alcune delle applicazioni includono l'utilizzo di queste cellule come biomarcatore per identificare i pazienti con rischio cardiovascolare, trattamento delle malattie ischemiche, e costrutti di creazione valvola vascolare e cuore tessutale.

Video: 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.

Induzione diretta di cellule staminali neurali umane da cellule periferiche del sangue emopoietiche progenitrici

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

Ricerca

Biologia dello sviluppo

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

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.

Isolamento di cellule murine embrionali Hemogenic endoteliali

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

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.

Isolare le cellule progenitrici Mesangiogenic (PPM) da midollo osseo umano

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

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.

Isolamento e caratterizzazione di cellule mesenchimali stromali dal Cordone ombelicale e placenta fetale

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

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Formazione di pellet condensato da cellule staminali pluripotenti indotte dal sangue

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Induzione di differenziazione endoteliale in cellule progenitrici cardiache in condizioni di basso del siero

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Isolamento e caratterizzazione di umani cellule staminali mesenchimali del cordone ombelicale-derivato da prematuri e neonati a termine

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

Generazione in Vitro di Somite derivati da umani indotta da cellule staminali pluripotenti indotte

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...