This work was supported by grants funded by the British Heart Foundation (BHF), Dinosaur Trust, McAlpine Foundation, Fondation Leducq, Fight for Sight, the Cambridge Biomedical Research Centre, National Institute of Health Research including (i) the BHF Oxbridge Centre of Regenerative Medicine [RM/13/3/30159], (ii) the BHF Cambridge Centre of Research Excellence, (iii) Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust and (iv) Papworth Hospital NHS Foundation Trust, and supported the Cambridge NIHR BRC Cell Phenotyping Hub. MLO is funded by a BHF Intermediate Fellowship. FNK is funded by a BHF PhD Studentship.

Uno schema del protocollo BOEC generazione è mostrato in Figura 1. 1. Raccolta di sangue e di densità centrifugazione in gradiente <ol><li> Per ciascun donatore, aggiungere 3 ml di citrato di sodio in ciascuna delle due provette da centrifuga 50 ml coniche. Raccogliere 60 ml di sangue per via venosa e aggiungere 30 ml di ogni provetta. Capovolgere delicatamente 2-3 volte per mescolare. Utilizzare non più di 40 ml di sangue nel protocollo, con volumi di citrato adeguati di conseguenza. NOTA: E &#39;essenziale che i campioni di sangue sono trattati entro 2 ore di raccolta. Ritardata elaborazione dei risultati del sangue in una marcata riduzione della resa colonia escrescenza. </li><li> Preparare le provette coniche nuove contenenti gradiente di densità media centrifugazione dal primo capovolgendo il flacone diverse volte per mescolare. In una cappa sterile, aggiungere 15 ml di gradiente di densità media centrifugazione per tubo da 50 ml (1 provetta per ogni 10 ml di sangue, 6 tubi per donatore). </li><li> Diluire il sangue 1: 1 con il fosfato di Dulbecco-Buffered Saline (DPBS). Inclinare il tubo contenente il mezzo di centrifugazione in gradiente di densità per un angolo di 20 ° con il piano di lavoro. Usando una pipetta aiuti e 25 ml pipetta sierologica, strato lentamente 21 ml di sangue diluito sopra del mezzo aggiungendo gradualmente sangue lungo la parete del tubo inclinato. NOTA: Ciò minimizzerà miscelazione del sangue con lo strato gradiente di densità e produrrà un buffy coat stretto, nonché un rendimento migliorato. </li><li> Centrifugare i campioni a 400 xg per 35 minuti a temperatura ambiente con l&#39;acceleratore e il freno. </li><li> Durante questo periodo la centrifugazione, iniziare il rivestimento collagene dettagliato nei passi 2.1 - 2.4. Assicurarsi che questi passaggi sono stati completati prima di procedere al punto 3.1. </li></ol> 2. Il collagene Rivestimento di T-75 Flask e preparazione del terreno di coltura NOTA: Eseguire le seguenti operazioni in una cappa di coltura cellulare. <ol><li> Preparare una soluzione di collagene / ml 50 mg diluendo magazzino TyPe I collagene in 10 ml di acido acetico 0.02M. Esempio: per il collagene magazzino ad una concentrazione di 4,05 mg / ml, aggiungere 123,5 ml di collagene per 10 ml di acido acetico 0,02 M. Sospensione collagene filtro sterile prima dell&#39;uso. </li><li> Usando una pipetta sierologica, aggiungere 7,5 ml di soluzione di collagene in un pallone T-75 coltura cellulare. Questo volume dà 5 mg collagene / cm 2 (o 375 mg / beuta). Coat pallone per 1 ora a RT. </li><li> Preparare il terreno di crescita endoteliale utilizzato per la generazione BOEC completandola medio basale endoteliale con il fattore di crescita integrazioni fornite dal produttore. Aggiungere tutti gli integratori, ma non aggiungere siero. Per preparare 15 ml di terreno BOEC generazione, aggiungere 2,5 ml di siero bovino fetale definita (FBS) a 12,5 ml di terreno di crescita endoteliale contenente il fattore di crescita supplementi. </li><li> Procedere con la procedura descritta nella sezione 3. Dopo il periodo di rivestimento 1 ora, aspirare la soluzione di collagene e acido acetico lavare via residuale pipettandoin 10 ml DPBS. Aspirare fuori DPBS e ripetere l&#39;operazione di lavaggio ancora una volta. Se la sospensione cellulare ottenuta durante la fase 3.3 non è pronto per la placcatura in questo momento, aggiungere 5 ml di mezzo BOEC generazione al pallone per mantenere il rivestimento collagene da essiccazione-out. </li></ol> 3. Raccolta e placcatura di PBMNCs <ol><li> Dopo la centrifugazione in gradiente di densità descritto al punto 1.4, raccogliere con cura lo strato buffy coat con una pipetta di trasferimento sterile in plastica. Raccolta di buffy coat dovrebbe produrre circa 20 ml di sospensione cellulare e plasma da ciascuna provetta. Evitare di trasferire il mezzo gradiente di densità. </li><li> Diluire la sospensione di cellule mononucleate 1: 1 in DPBS e miscelare. Centrifugare a temperatura ambiente per 20 min a 300 xg con freno e dell&#39;acceleratore al massimo. </li><li> Dopo la centrifugazione, aspirare il surnatante e risospendere le cellule aggiungendo 1 ml di terreno BOEC generazione in ogni pellet e pipettando su e giù ripetutamente. Cella Pool sospensioni und rabboccare volume totale di 10 ml con il mezzo rimanente. </li><li> Per ottenere una stima del numero totale delle cellule, campione 10 ml di sospensione cellulare e diluire 50x con 490 ml di soluzione Turk, che la lisi dei globuli rossi. Contare un campione di 10 microlitri di sospensione cellulare diluita utilizzando un emocitometro. NOTA: 60 ml di sangue dovrebbe produrre circa 100-150 x 10 6 cellule bianche del sangue per la placcatura. </li><li> Piastra sospensione intera cella in un unico, collagene rivestite T-75 pallone, rabboccare il volume medio 15mL / beuta e coltura a 37 ° C in atmosfera umidificata contenente il 5% di CO 2. Le cellule placcati in questo momento rappresentano il passaggio 0. NOTA: È possibile congelare le PBMNCs isolate prima dell&#39;isolamento BOEC per derivare le BOECs in un secondo tempo o per trasportare i PBMNCs ad un laboratorio diverso in cui possono essere generati i BOECs. Tuttavia, è importante notare che PBMNCs congelamento potrebbero ridurre l&#39;efficienza di isolamento BOEC. SSezione 8 ee sotto per il metodo. </li></ol> Colture cellulari 4. A lungo termine <ol><li> Cambiare media ogni 2 giorni con l&#39;aggiunta di 15 ml di mezzo fresco generazione BOEC (5: 1 miscela di cellule endoteliali mezzo di crescita e definito FBS). Per il fine settimana, i cambiamenti medi possono essere ritardate per ogni 3 giorni. NOTA: le colonie conseguenza dovrebbe apparire tra 7 e 14 giorni di coltura. </li><li> Monitorare il pallone di coltura BOEC nei giorni 7-14 per la comparsa di colonie escrescenza. Identificare le colonie come gruppi circolari di cellule che presentano un classico ciottoli morfologia endoteliale. </li><li> Una volta che una colonia o più colonie sono identificate nel pallone, continuare con i cambiamenti medi e permettono colonie di crescere a circa 1000 a 2000 cellule per colonia prima passaging. Determinare il numero di cellule per colonia attraverso una stima visiva approssimativa. NOTA: Come guida, si è soliti passaggio colonie escrescenza iniziali Una settimana dopo l&#39;identificazione della prima colonia conseguenza. </li> &lt;li&gt; cellule Passage di risciacquo fiaschi due volte con 10 ml di DPBS. Aggiungere 5 ml di 1X tripsina-EDTA e incubare in un incubatore a 37 ° per 5 minuti. Dopo 5 minuti, neutralizzare tripsina con 10 ml di terreno (contenente FBS) e portare le cellule in sospensione con pipettaggio ripetuto. </li><li> Sospensione Centrifugare a 300 xg per 5 min, risospendere in 15 ml di mezzo fresco generazione BOEC e piastra intera sospensione cellulare in un pallone nuovo T-75 (che rappresenta il passaggio 1, P1). Non è richiesto alcun rivestimento collagene. </li><li> Continuare cambiamenti terreno come descritto sopra finché le cellule sono confluenti (circa 3-5 x 10 6 cellule per pallone). Una volta confluenti, cellule di passaggio come descritto al punto 4.3. NOTA: Dal passaggio 2 in poi, le cellule richiedono non più di media BOEC generazione e può essere coltivato in endoteliali terreno di crescita cellulare e il 10% di serie FBS inattivato al calore. Rivestimento Il collagene può essere usato come un passo opzionale andando avanti, in quanto non vi sono prove che suggeriscono che la presenza di collagene può migliorare prolifer BOECtassi ation. </li><li> Per preservare le passaging, piatto non meno di 750.000 cellule per T-75 pallone come la densità di cellule basse possono causare i BOECs per fermare la proliferazione. </li></ol> 5. Congelamento e culture scongelamento BOEC <ol><li> Trypsinize cellule come descritto nella sezione 4.3 e centrifugare a 300 xg per 5 min. Aspirare il surnatante e risospendere in 10 ml di terreno. Ciò non richiede endoteliale terreno di crescita cellulare; DMEM con 10% FBS standard, sarà sufficiente. Raccogliere un campione di 10 microlitri di sospensione cellulare per eseguire un numero di cellule utilizzando un emocitometro manuale. </li><li> Ancora centrifuga e risospendere a 2x10 6 cellule / ml in mezzo di crioconservazione ghiacciata (40% DMEM con il 50% FBS e il 10% DMSO). Aggiungere 0,5 ml (10 6 celle) per ogni fiala, posto fiale in un ghiaccio isopropanolo freddo nave crioconservazione e posto in una -80 ° C freezer per almeno 2 ore prima del trasferimento di azoto liquido. Non lasciare celle a -80 ° C per più di 24 ore. </li&gt; <li> Per scongelare le cellule, aggiungere 10 ml di terreno pre-riscaldato a un 15 ml provetta da centrifuga conica. NOTA: Per scongelare fuori passaggio 1 cellule, scongelare in endoteliali terreno di coltura cellulare integrato con il 20% FBS definito per i primi 2 giorni dopo lo scongelamento. Questo porta ad un isolamento delle cellule più stabile per il futuro. </li><li> Rimuovere le cellule di azoto liquido e disgelo in un bagno d&#39;acqua a 37 ° con agitazione fino a quando solo un piccolo cristallo di ghiaccio è rimasto nel flaconcino. Aggiungere il contenuto della fiala goccia a goccia nella provetta da centrifuga conica e centrifugare a 300 xg per 5 min. </li><li> Aspirare il surnatante e risospendere pellet cellulare in 10 ml EGM-2MV + 10% FBS. Aggiungi a pallone T-75 e rabboccare medio a 15 ml. </li></ol> 6. Caratterizzazione di BOECs mediante citometria di flusso <ol><li> Una volta che le colonie BOEC cui alla Sezione 4 sono stati autorizzati a espandersi, trypsinize cellule come descritto nel paragrafo 4.4 e risospendere ad una concentrazione di 10 6 cellule per ml in tampone colorazione (DPBS ingegnoh 2% FBS e 2 mM EDTA). </li><li> Combina 10 5 cellule con anticorpi coniugati a fluorocromi diretti contro CD45, CD14, CD34, CD31 (uso tutto a 1:20 diluizione) o VEGFR2 (diluito 1:10) (vedi Tabella 1 per i dettagli). Incubare per 30 minuti a 4 ° C al buio. </li><li> Lavare le cellule con 1 ml di tampone di colorazione e centrifugare a 300 xg per 5 min. Aspirare il surnatante e risospendere in 400 microlitri di buffer di colorazione per l&#39;analisi. Mantenere cellule a 4 ° C e al riparo dalla luce prima dell&#39;analisi. </li><li> Eseguire l&#39;analisi di citometria su un citometro attrezzato per rilevare gli anticorpi coniugati fluorescenza utilizzati nel test. </li><li> Utilizzare un campione BOEC macchia per identificare la popolazione di cellule sul citometro regolando avanti e tensioni scatter laterale, così come le tensioni per i canali FITC e APC. </li><li> Determinare gating soglie utilizzando cellule isotipo colorate per ogni fluorocromo. Valutare positività per ogni marcatore superficie cellulare basato sulla Presence di uno spostamento picco dell&#39;intensità di fluorescenza contro controllo isotipo per il fluorocromo analizzato. </li></ol> 7. Caratterizzazione di BOECs da immunofluorescente Microscopia <ol><li> BOECs piatto in un pozzo 24, a fondo piatto piastra di coltura tissutale senza vetrini in una densità di 5 x 10 4 cellule in 500 ml di endoteliale terreno di crescita delle cellule + 10%-inattivato con il calore FBS per bene e lasciare ad aderire e crescere a 37 ° C, 5% CO 2 fino cellule coprire almeno il 70-80% della superficie di ciascun pozzetto (stima confluenza mediante ispezione visiva sotto una microsope luce). </li><li> Lavare le cellule in ciascun pozzetto per 5 min con 1 ml di DPBS. </li><li> Fissare cellule O / N a 4 ° C con 500 ml di soluzione di paraformaldeide 4% in DPBS. </li><li> Lavare le cellule per 3 x 5 min con 1 ml di DPBS per pozzetto. Aggiungere e rimuovere i DPBS utilizzando una pipetta e un aspiratore, rispettivamente. </li><li> Permeabilize cellule da 3 x 10 minuti con 0,2% polisorbato 20 in DPBS. </li&gt; <li> Incubare le cellule per 1 ora a RT in tampone di bloccaggio contenente 10% FBS in DPBS. </li><li> Cellule Stain a 4 ° CO / N in 300 ml di tampone di diluizione anticorpo (0,1% di albumina di siero bovino in DPBS) contenente anticorpi primari diretti contro CD34 (10 mcg / ml), CD144 (1: 300) o vWF (1: 250) . Vedere la Tabella 1 per tutti i dettagli. </li><li> Lavare gli anticorpi primari non legati per 5 x 10 min con DPBS. </li><li> Incubare le cellule per 1 ora a RT con l&#39;anticorpo secondario fluorescenza marcata appropriata, come indicato nella tabella 1 (tutto a 1: 200). </li><li> Lavare anticorpi secondari non legati per 5 x 10 min con DPBS. </li><li> Incubare le cellule con 1 mg / ml 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) in DPBS per 10 minuti a RT. </li><li> Lavare le cellule con DPBS per ulteriori 3 x 5 min. </li><li> Celle di immagine di ingrandimento 100x utilizzando un microscopio invertito dotato di una sorgente di luce esterna per l&#39;eccitazione di fluorescenza, e catturare immagini con un allegarefotocamera digitale cato per la successiva analisi offline. </li></ol> 8. gelo e disgelo PBMNCs <ol><li> Al termine della fase 3.2, dopo centrifugazione, aspirare il terreno e interrompere manualmente il pellet cellulare con pipettando ripetutamente su e giù con una punta della pipetta P1000. Aggiungere 1 ml di mezzo di congelamento, siero 90% e il 10% DMSO, avendo cura di mescolare delicatamente per ottenere una sospensione di cellule. Trasferire la sospensione cellulare per un esageratamente e congelare e scongelare come descritto nella Sezione 5. </li></ol>

<ol>
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The authors have nothing to disclose.

We present a detailed protocol that allows for the robust and efficient derivation of BOECs from adult peripheral blood mononuclear cells (PBMNCs). Our protocol includes two important refinements that represent advances on previous methods of BOEC isolation.14-16 These include the absence of heparin in the initial PBMNC culture medium and the use of defined, embryonic stem cell-qualified serum. This latter refinement is of particular importance. Embryonic stem cell (ESC)-qualified serum is a more consistent grade of serum and, although it is not known yet what component(s) are enriched in the serum that benefit BOEC isolation, the impact of this defined serum on the efficiency of BOEC generation is clear in our hands. In addition, we have also had success in isolating BOECs using human serum, thereby allowing for the generation of BOECs for clinical translation. In our hands, this refined protocol results in the successful isolation of stable BOEC cultures from greater than 90% of donors, making it one of the most reliable BOEC generation methods reported thus far. Although the use of particular sera is critical to BOEC generation, it also represents a primary limitation of the current protocol. Future improvements to the technique could include the generation of these cells in serum-free, defined culture conditions.
Critical Steps in the protocol include processing blood samples as soon as possible after collection, complete harvesting of the buffy coat cells after density gradient centrifugation and the timely passaging of initial colonies from P0 to P1. This passaging step is critical to establishment of a stable isolation. Like other endothelial cells, BOECs appear to be very sensitive to plating density. If the plating density after passaging is too low, the BOECs will not proliferate. Conversely, if the colonies are allowed to become overconfluent before passaging, the cells will also cease to proliferate and have the tendency to convert into an elongated, mesenchymal cell phenotype. If few colonies appear from days 7 to 14, or if the colonies are small in size, troubleshooting can include increasing cell density by passaging P0 colonies into a T-25 flask instead of a T-75.
Once the technique is mastered, the resultant BOECs can be used in several applications, including in vitro studies of endothelial cell biology, disease modeling and drug screening, as well as in vivo cell transplantation therapies. An important consideration for the development of any cell therapy process is to use cells that are free from pathogenic mutations. We have previously shown that BOECs isolated using our protocol possess genomes that are free from copy number variations and are thus representative of the individual from which they were collected. In addition, we have also demonstrated that the majority of BOEC-derived iPSC lines are free from copy number variations.13 This contrasts with previous reports of copy number variation in fibroblast-derived iPSCs. To date, these cells remain the only iPSCs for which this degree of genomic fidelity has been reported. This feature is important for the field of iPSC biology and the use of iPSCs in disease modeling, drug screening and future cell transplantation therapies.

Fino a poco tempo, la generazione post-natale di nuovi vasi sanguigni è creduto avvenire esclusivamente attraverso un processo noto come angiogenesi, definito come il germogliare di nuovi vasi dalle cellule endoteliali dei vasi preesistenti. 1 Questo processo contrasta dalla vasculogenesi, o la formazione de novo di vasi sanguigni da progenitori endoteliali, che è stato pensato per manifesta esclusivamente durante l&#39;embriogenesi. 2 Tuttavia, studi più recenti hanno identificato e isolato cellule progenitrici endoteliali circolanti (EPC) nel sangue periferico di adulti. Queste cellule possiedono la capacità di differenziarsi in cellule endoteliali mature di cultura e si ritiene che partecipare a vasculogenesi postnatale. 3,4 Protocolli per l&#39;isolamento e l&#39;espansione di queste EPC tipicamente coinvolgono la coltura di cellule mononucleate del sangue periferico (PBMNCs) in supporti contenenti fattori di crescita endoteliali, tra cui vascular fattore di crescita endoteliale (VEGF) e le culture di crescita dei fibroblasti fattore-2. 5-8 EPC produrre una varietà di tipi di cellule drammaticamente diversi. Culture iniziali (&lt;7 giorni) sono dominati da un tipo di cellula monocitica, noto in letteratura come &quot;early&quot; EPC. Nonostante il loro nome, queste cellule esprimono il CD14 marcatore monociti, sono negativi per il CD34 marcatore progenitore ed esprimono livelli solo minimi della classica endoteliale marcatori CD31 e VEGF recettore 2 (VEGFR2). 5 coltura continua dà luogo ad una popolazione secondaria di cellule, noto come EPC fine escrescenza o cellule endoteliali escrescenza del sangue (BOECs), che appaiono come colonie discreti di cellule endoteliali-like. A differenza dei primi monocitiche EPC, BOECs, che sono stati anche chiamati formanti colonie cellule endoteliali (ECFCs), cellule endoteliali escrescenza o cellule endoteliali tardo-escrescenza, presentano la morfologia ciottoli che è tipica di monostrati cellule endoteliali e sono molto simili a marcatore di superficie5 e l&#39;espressione genica 9 a maturare cellule endoteliali. La generazione di cellule endoteliali-simili da sangue periferico offre numerosi vantaggi, in particolare per lo studio della disfunzione delle cellule endoteliali associata a disturbi vascolari quali ipertensione arteriosa polmonare (PAH) 10 o malattia di von Willebrand. 11 Prima della disponibilità di BOECs, endoteliale cellule potrebbero essere derivate solo da organi espiantati al momento della morte o trapianto di organi, o isolate dalla vena ombelicale al momento della nascita. Questa ridotta disponibilità rappresentato una seria limitazione alla comprensione della biologia delle cellule endoteliali di pazienti con disturbi cardiovascolari, nonché le interazioni tra cellule endoteliali e sia cellule del sangue o cellule murali. Inoltre, isolando e coltivando una popolazione pura di cellule endoteliali da queste fonti è tecnicamente difficile e le cellule derivate da questi metodi presentano solo un limited capacità proliferativa. BOECs quindi offrire un surrogato valido per l&#39;isolamento e la coltura di cellule endoteliali primarie derivate paziente. Oltre alle loro applicazioni in vitro, BOECs sono anche potenzialmente utili in autologhe terapie di trapianto cellulare. Queste applicazioni comprendono sia il trapianto di cellule endoteliali di promuovere la neovascolarizzazione (vedi 12 e riferimenti ivi), nonché la generazione di cellule staminali pluripotenti indotte (iPSCs). 13 iPSCs BOEC derivati ​​possono essere utilizzate per modellare la malattia e offrono un potenziale immenso come partenza materiale per terapie cellulari autologhe. BOECs riprogrammare più velocemente e con un rendimento superiore fibroblasti cutanei. Inoltre, BOECs permettono anche per la generazione di iPSCs che siano privi di anomalie del cariotipo, che è una caratteristica essenziale di qualsiasi tecnologia che sarà adatto per applicazioni traslazionali. La capacità di generare iPSCs da un campione di sangue del paziente unaLSO elimina la necessità di una biopsia cutanea e la generazione di fibroblasti cutanei, facilitando in tal modo la generazione di cellule da pazienti con disturbi guarigione delle ferite, o molto giovane. Il protocollo di seguito dettagliato, approvato e condotto in conformità con le linee guida del Comitato Research Service Etico Nazionale (Est Inghilterra), fornisce un metodo semplice e affidabile per la generazione di BOECs con efficienza superiore al 90% da un volume relativamente piccolo (60 ml) di sangue periferico. Queste cellule sono altamente proliferative e possono essere diversi passaggi più volte, permettendo la generazione di centinaia di milioni di cellule da un singolo campione di sangue.

<table>
	<tbody>
		<tr>
			<td colspan="4">For blood collection</td>
		</tr>
		<tr>
			<td>60 mL syringe with luer-lok tip</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>19G Surflo Winged Infusion Set</td>
			<td>Terumo</td>
			<td>SV-19BL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>StarLab</td>
			<td>E1450</td>
			<td>2 per donor</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Martindale Pharmaceuticals</td>
			<td>270541</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For buffy coat isolation</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile wrapped plastic transfer pipettes</td>
			<td>Appleton Woods</td>
			<td>KC231</td>
			<td></td>
		</tr>
		<tr>
			<td>Turk&#8217;s Solution</td>
			<td>Millipore</td>
			<td>1.093E+09</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For cell culture, passaging and freezing cells</td>
		</tr>
		<tr>
			<td>Type 1 Collagen (derived from rat tail)</td>
			<td>BD Biosciences</td>
			<td>35-4236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.02M Acetic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>prepared in reagent grade water</td>
		</tr>
		<tr>
			<td>Endothelial Growth Medium-2MV 
			(containing Bullet Kit, but not serum)</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Note: It is essential that the medium does not contain heparin.&#160;&#160; 
			Do not use EGM-2.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (U.S.), Defined</td>
			<td>Hyclone</td>
			<td>SH30070</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Trypsin EDTA</td>
			<td>Gibco</td>
			<td>T4174</td>
			<td>Dilute to 1x in PBS prior to use</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>C1562</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For flow cytometric characterization</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD14</td>
			<td>BD Biosciences</td>
			<td>555397</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD31</td>
			<td>BD Biosciences</td>
			<td>555445</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-human CD34</td>
			<td>BD Biosciences</td>
			<td>555824</td>
			<td>Mouse IgG1k, Clone: 581/CD34 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>560976</td>
			<td>Mouse IgG1k, Clone: HI30 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>&#160;APC-conjugated mouse anti-human VEGFR2</td>
			<td>R&#38;D Systems</td>
			<td>FAB357A</td>
			<td>Mouse IgG1, Clone: 89106 
			Dilution: 1:10</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555751</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>R&#38;D Systems</td>
			<td>IC002A 
			Dilution: 1:10</td>
			<td>Clone: 11711</td>
		</tr>
		<tr>
			<td>EDTA, 0.5M solution</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For immunofluorescent microscopy&#160;</td>
		</tr>
		<tr>
			<td>Corning Costar 24-well tissue culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD34 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB72271</td>
			<td>Clone 756510, IgG1, use at 10 &#956;g/ml</td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-human VE-cadherin (CD144)</td>
			<td>R&#38;D Systems</td>
			<td>AF938</td>
			<td>Antigen affinity- purified IgG, use at 1:300</td>
		</tr>
		<tr>
			<td>Monoclonal rabbit anti-human Von Willebrand Factor (vWF)</td>
			<td>Abcam</td>
			<td>ab154193</td>
			<td>Clone EPSISR15, use at 1:250</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
			<td>Polyclonal, 2 mg/ml, use at 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11055</td>
			<td>Polyclonal, 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 568 conjugate</td>
			<td>Life Technologies</td>
			<td>A-10042</td>
			<td>Polyclonal,&#160; 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use at 1 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

generation and culture of blood outgrowth endothelial cells from human peripheral blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

Isolamento di buffy cellule mononucleate cappotto da 60 ml di sangue produce tipicamente 100-150x10 6 totali globuli bianchi. Quando placcato in un&#39;unica T-75 pallone, il gran numero di cellule in sospensione cellulare non lisati rende difficile risolvere singole cellule aderenti mediante microscopia in campo chiaro. Cambiamenti medi ripetute provocano il passaggio di cellule non aderenti e consentono la visualizzazione della popolazione aderente monocitica &quot;primi&quot; EPC. Al giorno 7, il T-75 conterrà circa 7x10 6 di queste cellule aderenti allungate. Dal giorno 7-14, colonie di BOECs dovrebbero apparire all&#39;interno del pallone (Figura 2A). Colonies prima appaiono come 3 a 5 celle adiacenti. Numero colonia conseguenza può variare da 1 a 10 colonie per 60 ml di sangue. Generalmente, i donatori più giovani (cioè, 20-25 anni) tendono a produrre un maggior numero di colonie escrescenza, che appaiono anche in precedenza in coltura.BOECs proliferano da questo gruppo centrale di cellule di formare colonie circolari composti da diverse centinaia di cellule. Cellule all&#39;interno di queste colonie presentano un classico ciottoli morfologia endoteliale. È preferibile passaggio colonie iniziali quando contengono circa 1000 cellule per colonia. Colonie escrescenza tipicamente raggiungono queste dimensioni 7 giorni dopo il loro aspetto originario nella cultura. Una volta che le colonie originali sono diversi passaggi in un pallone nuovo T-75, dovrebbero proliferare di formare un monostrato confluente (3-5x10 6 cellule per pallone) entro 5 giorni o meno (Figura 2B). In una piccola percentuale di isolamenti (&lt;10%), le colonie escrescenza non si presentano, o non proliferano sufficientemente seguendo questo primo passo passaging. BOECs che presentano bassa proliferazione dopo il loro primo passaging raramente vanno a diventare isolamenti BOEC stabili e spesso smettere di proliferare entro due o tre passaggi. Il monociticaprimi EPC contenuti all&#39;interno delle culture BOEC sono non proliferativa e sono tipicamente cancellati dalle culture in 1-2 passaggi. Liquidazione di questi primi cellule è dovuta alla morte cellulare, il fallimento dei primi cellule di ri-aderire dopo passaging e diluizione dei non proliferative primi EPC con passaging ripetuto. Passage celle 1 possono sia essere diversi passaggi ulteriormente nella cultura o congelati giù per un uso successivo. Una volta generato un isolamento stabile, le cellule possono essere diversi passaggi ad una velocità di 1 boccetta confluenti a 3-5 nuovi T-75 beute fino passaggio 8 o 9 prima di diventare quiescente. Anche in questo caso, la densità delle cellule è fondamentale per tutta la cultura. Nella nostra esperienza, non meno di 750.000 cellule devono essere placcato in un pallone T-75, come la densità di cellule più bassi possono causare arresto della crescita. Nonostante l&#39;elevato potenziale proliferativo di BOECs, cellule devono anche non essere permesso di diventare overconfluent (es.,&gt; 5x10 6 cellule per pallone) e questo può causare Convers<html> ioni di BOECs ad un fenotipo non proliferativa. Proponiamo che ogni cellula di essere etichettato come un BOEC deve visualizzare superficie cellulare appropriato e colorazione intracellulare per i marcatori endoteliali. Caratterizzazione di BOECs può essere ottenuto mediante citometria di flusso (figura 3) o microscopia a fluorescenza (Figura 4), ​​dopo colorazione per marcatori tipici delle cellule endoteliali. BOECs sono positivi per il CD31 marcatori di superficie endoteliale e VEGFR2 e sono negativi per il CD14 marcatore monociti e il pan-leucociti marcatore CD45. BOECs hai tutti anche i corpi di Weibel-Palade e quindi esprimere von Willebrand (vWF) come discreta, puntata colorazione citoplasmatica. A differenza di altri tipi di cellule endoteliali mature, come arteria polmonare o cellule endoteliali aortiche, BOECs esprimono anche il progenitore e l&#39;attivazione marcatore CD34 sulla loro superficie. ftp_upload / 53384 / 53384fig1.jpg &quot;/&gt; Figura 1. Schema di protocollo BOEC generazione. Cellule mononucleate del sangue periferico sono isolati dal sangue venoso dal gradiente di densità centrifugazione e coltivate su piastre di collagene rivestite con terreno di crescita endoteliale contenenti definiti FBS. Colonie BOEC compaiono entro 7-14 giorni di coltura. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53384/53384fig2.jpg" /> Figura 2. Immagini Brightfield di culture BOEC rappresentativi. (A) colonie escrescenza appaiono in culture tra i giorni 7 e 14. Le colonie presenti collezioni di cellule endoteliali-simili, che sono disposti in un monostrato di ciottoli e proliferano radialmente fuori da un punto centrale. Intorno le colonie escrescenza sono mono aderenticellule cytic che costituiscono la stragrande maggioranza delle cellule nelle colture precoci. Queste cellule, precedentemente descritte come &quot;presto&quot; le cellule progenitrici endoteliali, hanno una morfologia fuso-like e esprimono il marcatore CD14 monocitiche. (B) A seguito passaging, i BOECs altamente proliferative prendere in consegna le culture, come le cellule monocitiche non proliferative o muoiono o non riescono a riattaccare dopo passaging. Scala bar 250μm. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53384/53384fig3.jpg" /> Figura 3. Caratterizzazione di BOECs mediante citometria di flusso. BOECs stati tripsinizzate e colorati con gli anticorpi di controllo isotipo fluorescente-coniugati (gray picco pieno) o anticorpi diretti contro marcatori di superficie specifici (linea rossa). Marcatori di superficie per la caratterizzazione di citometriaincludono i marcatori ematopoietici CD45 e CD14, i marcatori endoteliali CD31 e VEGFR2 e la progentior e marcatore endoteliale CD34. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53384/53384fig4.jpg" /> Figura 4. immunofluorescenza di BOECs per i marcatori delle cellule endoteliali. Immunofluorescenza immagini rappresentative dei BOECs immunostained con anticorpi diretti contro endoteliali marcatore CD144 superficie cellulare (VE-caderina, pannello superiore a destra) e la glicoproteina sangue Fattore di von Willebrand (vWF, in basso pannello di destra) . Pannelli corrispondenti mostrano colorazione DAPI nucleare sono mostrati a sinistra. Scala della barra 50 micron. per CD144. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig4large.jpg" target="_blank">Cliccate qui per vedere una più grande</a>versione di questa figura.

Watch this Scientific Journal Video about Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood at JoVE.com

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Generazione e Cultura Sangue escrescenza endoteliali cellule da sangue periferico umano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

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18.8K Views.  University of Cambridge. This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Video: Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Arricchimento di cellule NK dal sangue umano con il kit RosetteSep Technologies StemCell

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Ricerca

Biologia

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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.

Caratterizzazione fenotipica e funzionale delle cellule endoteliali Colony Forming derivato dal sangue del cordone ombelicale umano

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

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Generazione di cellule pluripotenti indotte umane staminali da sangue periferico mediante il STEMCCA lentivirale Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Purificazione e microRNA profiling di Exosomes derivati ​​dal sangue e cultura dei media

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Generazione efficiente delle cellule iPS da Blood Utilizzando episomi e HDAC inibitori

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

A due fotoni Imaging di Intracellulare Ca<sup&gt; 2+</sup&gt; Manipolazione e l&#39;ossido nitrico Produzione in endoteliale e della muscolatura liscia cellule di un isolato Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Le proteine ​​immunofluorescenza di endogeni ed esogeni Centromere-cinetocoro

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

Isolamento e caratterizzazione di microvescicole da sangue periferico

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Un saggio di taglio uniforme per piastrine umane e recettori superficiali delle cellule tramite viscometria conica

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...