Gli autori ringraziano il Dott. Katharina Schallmoser per la fornitura di pHPL e Margaretha Frühwirth e Daniela Thaler per un&#39;eccellente assistenza tecnica.

A.) ECFC ISOLATON GIORNO 1: <ol><li> Prima di iniziare con l&#39;esperimento preparare la cultura della crescita delle cellule endoteliali medio a medio-2 (EGM-2). Supplemento 500ml endoteliale basale medio-2 con eparina (1 ml di 1000 U / ml), 5 ml soluzione 100x di penicillina / streptomicina (tutti Sigma, St. Louis, MO, <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamina (concentrazione 2 mM finale ), e 50 ml pool lisato piastrinico umano (pHPL). Quindi aggiungere 0,2 ml di idrocortisone, 2 ml hFGF-B, 0,5 ml di VEGF, EGF 0,5 ml, 0,5 ml di IGF-1 e 0,5 ml di acido ascorbico (fornito come SingleQuots, tutti Lonza, Walkersville, MD; <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience. com /</a> ). Sterile filtrato del mezzo attraverso un m-pori 0,2 dimensioni 500 ml vuoto filtro Millipore. Dal pHPL forme coaguli piccoli potrebbe essere necessario due filtri per un mezzo. Preriscaldare filtrata EGM-2 a 37 ° C in un bagno d&#39;acqua </li><li> Raccogliere i campioni di sangue da disegno 5 ml di sangue periferico umano adulto dalla vena cubitale in una 6ml senza conservanti sodio-eparina tubo sangue vacutainer. Si dovrebbe trattare i campioni di sangue entro un massimo di due ore. </li><li> Iniziate preparando una compressa da 75 cm 2 (T75; Costar) recipiente con un tappo di sfiato, pipetta da 25 ml e due 5 pipette ml in un banco a flusso laminare. </li><li> Pre-compilati 15 ml preriscaldata integrato EGM-2 nel pallone con la pipetta da 25 ml. Ora aprite la fiala di sangue e campioni tutti e 5 ml con la pipetta 5 ml nel pallone. Sciacquare la fiala vuota di sangue con 5 ml di ulteriori EGM-2 con la seconda pipetta da 5 ml. Il volume totale entro il T75 è di 25 ml. Chiudere il ventilato-cap del pallone e mettere in termostato a 37 ° C, 5% CO 2 e il 95% di umidità per 24 ore. </li></ol> 2 ° GIORNO: <ol><li> Dopo circa 24 ore (durante la notte) è necessario rimuovere il medio impasto sangue per liberarsi di cellule non aderenti. Per questo preparare due pipette 25 ml e tre 5 pipette ml nel flusso laminare, pre-riscaldare 20 ml di integrare EGM-2 (preparato il giorno precedente) e 30 ml di PBS a 37 ° C in un bagno d&#39;acqua. </li><li> Rimuovere il pallone con il campione dal termostato e rimuovere tutti i surnatante con una pipetta 25 ml. Fare attenzione a non graffiare la superficie pallone evitare di danneggiare qualsiasi potenziale colonia. Lavare la superficie interna delle cellule fiasco aderenza tre volte con delicatezza l&#39;aggiunta di 10 ml di pre-riscaldato PBS e inclinando il pallone da un lato all&#39;altro. Rimuovere la PBS immediatamente ogni volta e scartare. </li><li> Aggiungere 20 ml di fresca pre-riscaldato EGM-2 al pallone e rimetterlo a dell&#39;incubatore. Questo processo deve essere eseguita il più veloce e gentile possibile, per le cellule collegate non a staccarsi. ECFCs staccare facilmente se sono tenuti in PBS o a temperatura ambiente per lungo. </li><li> Schermo attraverso il pallone sistematicamente ogni due giorni a partire da due giorni con un basso ingrandimento di un microscopio ottico in cerca di escrescenza ECFC. Quando una colonia si trova segno sulla parte superiore sul lato esterno del pallone che indica la posizione della colonia. Si noti che il flaks non dovrebbe essere al di fuori dell&#39;incubatore per più di 5 minuti. </li></ol> GIORNO 5: <ol><li> Il quinto giorno dopo la semina effettuare un cambiamento completo di media per rimuovere le cellule non aderenti. A tal fine pre-riscaldare 20 ml di EGM-2 a 37 ° C in un bagno d&#39;acqua e preparare due pipette 25 ml. </li><li> Rimuovere il terreno completo con una pipetta 25 ml e aggiungere fresco, pre-riscaldata EGM-2 completate, non appena possibile. Le cellule non devono essere lasciati senza prodotto per più di un minuto per evitare l&#39;essiccamento. Mettere nuovamente dentro l&#39;incubatrice e ripetere lo screening del pallone tutti i giorni. </li><li> Nutrire le cellule, sostituendo il 30% dei media da parte fresca integrato EGM-2 volte alla settimana. </li></ol> GIORNO 14-28: <ol><li> Le colonie tipiche con morfologia ciottoli dovrebbe apparire intorno al giorno 12. Segna la parte superiore esterna del pallone ad indicare la posizione di ogni colonia. Lasciare le colonie a crescere fino a 28 giorni a meno che le singole cellule iniziano a staccarsi. Una volta, le colonie principali sono stati identificati possono essere raccolto tra i giorni 14 e 28, a seconda delle loro dimensioni. </li></ol> B.) ECFC VENDEMMIA <ol><li> Preriscaldare 5 ml 0,05% tripsina / EDTA, 20 ml di PBS e 5ml fresca integrato EGM-2. </li><li> Rimuovere completamente medio dal pallone in un tubo Falcon (fare attenzione a non toccare la superficie del pallone per evitare di danneggiare qualsiasi colonia potenziali) e lavare accuratamente le cellule con 10 ml di PBS preriscaldata. </li><li> Aggiungere 5 ml di tripsina / EDTA e incubare per 5 minuti in incubatrice. Le cellule non devono essere esposti a tripsina per più di 7 minuti dal momento che diventa tossica. Accuratamente toccando i lati del pallone aiuta le cellule a staccarsi. Spegnere l&#39;attività della proteasi tripsina con l&#39;aggiunta di circa 10 ml del mezzo utilizzato conservato cultura. </li><li> Rimuovere sospensione cellulare in un 50 ml Falcon tubo. Sciacquare il pallone una volta con 10 ml di PBS, che è successivamente aggiunto alla sospensione cellulare raccolto. Centrifugare la sospensione cellulare a 300 g per 7 minuti a 4 ° C per raccogliere le cellule e rimuovere la tripsina. Eliminare il supernatante e risospendere il pellet cellulare immediatamente in 1 ml di PBS. Tenere su ghiaccio. </li><li> Determinare il numero di cellule, come descritto in un protocollo di JOVE di R. Ricardo e K. Phelani 5. </li></ol> C.) ECFC ESPANSIONE <ol><li> Supplemento 500 ml EGM-2, come descritto in A 1.1). Pre-riscaldamento a 37 ° C in un bagno d&#39;acqua. </li><li> Per circa 2500 cm 2 di spazio cultura preparare una o 11 T220 (225 cm 2) o 15 T175 (175 cm 2) o uno a quattro strati fabbrica di cellule (CF-4; Nunc, Naperville, Illinois; <a href="http://www.nuncbrand.com/">http://www.nuncbrand . com</a> ). Inoltre avrete bisogno di due nuove sfiato-cap e un imbuto speciale sterile in panchina flusso laminare. </li><li> Per ECFCs seme con una densità di cellule di partenza di 100 c / cm 2 calcolo del numero delle cellule è necessaria. Per una CF-4 252.800 cellule sono risospese in 500 ml di fresca EGM-2 e versato nella CF-4 con l&#39;imbuto sterile. Chiudere uno sfogo con lo sfiato-cap e inclinare il CF-4 da un lato longitudine, consentendo una distribuzione uniforme del mezzo tra tutte e 4 le farine. Poi inclinare la camera posteriore e aprire il coperchio del sfiato-cap. Luogo CF-4 in incubatrice. </li><li> Cambia il 20% della media almeno una volta alla settimana fino CF-4 è confluenti. </li><li> Celle di raccolta, come descritto in B.) utilizzando 70 ml di tripsina / EDTA e circa 200 ml di PBS. </li></ol> RAPPRESENTANTE RISULTATI Usando questo metodo isolamento abbiamo osservato una formazione primaria colonia al giorno 12. In uno studio precedente erano in grado di recuperare una media di 4,0 ± 0,8 ECFC-colonie derivate da ogni ml di sangue periferico. ECFCs 4 ha mostrato un aspetto tipico di ciottoli. Dopo l&#39;espansione su larga scala, con una densità di semina di 100 cellule / cm 2, abbiamo ottenuto&gt; 1 x 10 8 cellule in un totale di circa 30 giorni da 3 maschi e 1 volontari di sesso femminile (età compresa tra i 26 e 50 anni). ECFC colto in queste condizioni hanno dimostrato di essere proliferativa, funzionale e stabile genomicamente. 4 Inoltre cellule possono essere conservate (nel 10% DMSO) in azoto liquido per un ulteriore uso. 3,4

<ol>
	<li>Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+endothelial+cells+in+sickle+cell+anemia.">Circulating activated endothelial cells in sickle cell anemia.</a> N Engl J Med. 337, 1584-1590 (1997).</li><li>Yoder, M. C., Mead, L. E., Prater, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.</a> Tissue Eng Part C Methods. 14, 185-196 (2008).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. , (2009).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. , (2008).</li></ol>

Questo isolamento romanzo e il metodo di espansione è un processo semplice ed efficiente che è più efficiente e meno che richiede tempo, evitando qualsiasi separazione cellulare (gradiente di densità non necessario) e meno costoso rispetto alle tecniche convenzionali. Usando contatto pHPL agli antigeni della specie bovina e putativo xenoimmunization successivo è esclusa. Durante il periodo di cultura piccoli coaguli si possono formare, causata dal pHPL. Sulla base della nostra esperienza di questi coaguli di non ef...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Endothelial basal Medium-2</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>500ml</td>
</tr>
<tr>
<td>EGM-2 SingleQuots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid</td>
</tr>
<tr>
<td>preservative-free Heparin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>&nbsp;</td>
<td>10U/ml per EGM-2</td>
</tr>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>2mM per EGM-2</td>
</tr>
<tr>
<td>Penicillin and Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>100U/mL Penicillin and 100&mu;g/mL Streptomycin per EGM-2</td>
</tr>
<tr>
<td>Trypsin/1mM EDTA </td>
<td>&nbsp;</td>
<td>Gibco Cell Culture, Invitrogen </td>
<td>&nbsp;</td>
<td>7ml for T75 flask 70ml for CF-4</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Four-layered cell factory (CF-4) </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Funnel</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T75cm2 culture flask (with vented cap)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 mL vacutainer tubes (preservative-free sodium heparin) </td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>preloaded with 108 IU/6ml of preservative-free sodium heparin</td>
</tr>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Squeezer</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0,2 pore size sterile filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>2x 500ml per EGM-2</td>
</tr>		</tbody>
		</table>
		</div>

isolation and large scale expansion of adult human endothelial colony forming progenitor cells

Questo lavoro introduce una nuova strategia di recupero per le cellule progenitrici endoteliali formanti colonia (ECFCs) da eparinato ma per il resto non manipolata adulto periferici umani all&#39;interno di una media di 12 giorni. Dopo l&#39;espansione su larga scala&gt; 1x10 8 ECFCs può essere ottenuto per ulteriori test. Vantaggiosamente utilizzando pHPL il contatto delle cellule umane con antigeni siero bovino può essere escluso. Mediante citometria di flusso ed immunoistochimica delle cellule isolate può essere caratterizzato come ECFC e la loro funzionalità in vitro per formare strutture di tipo vascolare possono essere testati in un test di Matrigel. Inoltre queste cellule possono essere iniettati per via sottocutanea in un modello murino di forma funzionale, vasi perfusi in vivo. Dopo un lungo periodo di espansione e la proliferazione crioconservazione, la funzione e la stabilità genomica sembrano essere conservati. 3,4 Questo animale-proteina isolamento libero e il metodo di espansione è facilmente applicabile a generare una grande quantità di ECFCs.

Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Cellule progenitrici endoteliali formanti colonia (ECFCs) sono uno strumento promettente per studiare l&#39;omeostasi vascolare e la riparazione.<sup&gt; 1,2</sup&gt; Questo documento introduce un nuovo metodo di siero animale libero per l&#39;isolamento e l&#39;espansione del ECFC da eparinizzato adulti sangue umano periferico con un pool di lisato piastrinico umano (pHPL) diminuendo il rischio di immunizzazione anti-bovino.

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

isolation-large-scale-expansion-adult-human-endothelial-colony

Research

JoVE Journal

Biology

10.6K Views.  Medical University of Graz, Austria. Cellule progenitrici endoteliali formanti colonia (ECFCs) sono uno strumento promettente per studiare l'omeostasi vascolare e la riparazione. 1,2 Questo documento introduce un nuovo metodo di siero animale libero per l'isolamento e l'espansione del ECFC da eparinizzato adulti sangue umano periferico con un pool di lisato piastrinico umano (pHPL) diminuendo il rischio di immunizzazione anti-bovino.

Video: Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

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

Isolamento di cellule umane endoteliali della vena ombelicale (HUVEC)

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

Ricerca

Biologia

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Su larga scala Schermi di librerie metagenomiche

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

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

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

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

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

Isolamento e di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

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

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolamento di cellule endoteliali valvolare

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Un dosaggio quantitativo di insulina che esprimono formante colonia Progenitori

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Isolamento di cellule CD133 + staminali del fegato per l&#39;espansione clonale

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento da colture primarie di fibroblasti umani

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...