Los autores agradecen a la Dra. Katharina Schallmoser para proporcionar pHPL y Frühwirth Margaretha y Thaler Daniela por su excelente asistencia técnica.

A.) ECFC ISOLATON DIA 1: <ol><li> Antes de comenzar con el experimento de preparar el cultivo de células endoteliales de crecimiento medio mediano-2 (EGM-2). Suplemento de 500 ml de medio base endotelial-2 con heparina (1 ml de 1000 U / ml), solución de 5 ml 100x de penicilina / estreptomicina (todos Sigma, St. Louis, MO; <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamina (2 mM concentración final de ), y 50 ml de lisado de plaquetas humanas agrupados (pHPL). A continuación, añadir 0,2 ml de hidrocortisona, 2 ml hFGF-B, 0,5 ml de VEGF, 0,5 ml EGF, 0,5 ml de IGF-1 y 0,5 ml de ácido ascórbico (siempre como SingleQuots, todos Lonza, Walkersville, MD; <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience. com /</a> ). Filtrado estéril el medio a través de 0,2 m de tamaño de poro 500 ml filtro de vacío Millipore. Desde pHPL forma coágulos pequeños es posible que tenga dos filtros de un medio. Pre-caliente filtrada EGM-2 a 37 ° C en un baño de agua </li><li> Recoger las muestras de sangre mediante la elaboración de 5 ml de sangre periférica humana adulta de la vena cubital en un conservante 6ml libre de sodio heparina tubo vacutainer de sangre. Se debe procesar las muestras de sangre en un plazo máximo de dos horas. </li><li> Comience por la preparación de una 75 cm 2 (T75, Costar) frasco con una tapa ranurada, una pipeta de 25 ml y dos pipetas de 5 ml en un banco de flujo laminar. </li><li> Pre-llenado 15 ml previamente calentada complementado EGM-2 en el matraz con la pipeta de 25 ml. Ahora abra el vial muestra de sangre y todos los de 5 ml con la pipeta de 5 ml en el matraz. Enjuague el frasco vacío de sangre con 5 ml de adicionales EGM-2 con el segundo 5 ml pipeta. El volumen total dentro de la T75 es de 25 ml. Cierre la tapa del respiradero de la botella y coloque en una incubadora a 37 ° C, 5% de CO 2 y el 95% de humedad durante 24 horas. </li></ol> DIA 2: <ol><li> Después de aproximadamente 24 horas (durante la noche) debe retirar la mezcla de sangre medio para deshacerse de las células no adherentes. Para esta preparación de dos pipetas de 25 ml y tres pipetas de 5 ml en el flujo laminar, pre-caliente 20 ml de complementarse EGM-2 (preparado el día anterior) y 30 ml de PBS a 37 ° C en un baño de agua. </li><li> Retirar el matraz con la muestra de la incubadora y eliminar todas las sobrenadante con una pipeta de 25 ml. Tenga cuidado de no rayar la superficie del frasco para evitar daños en cualquiera de sus colonias potencial. Lave la superficie interna de la célula frasco de adhesión tres veces suavemente añadiendo 10 ml de pre-calentado PBS y la inclinación de la botella de un lado a otro. Retire la PBS inmediatamente cada vez y desechar. </li><li> Añadir 20 ml de agua dulce precalentado EGM-2 en el matraz y colocarlo de nuevo en la incubadora. Este proceso debe realizarse lo más rápido y suave como sea posible para que las células adjunto no se puede desprender. ECFCs desprenderse fácilmente si se mantienen en PBS a temperatura ambiente o para la de largo plazo. </li><li> Pantalla a través del matraz sistemáticamente cada dos días a partir de dos días con un bajo aumento de un microscopio de luz en busca de fruto ECFC. Cuando una colonia se encuentra la marca en la parte superior de la parte externa de la botella que indica la posición de la colonia. Tenga en cuenta que el matraz no debe estar fuera de la incubadora por más de 5 minutos. </li></ol> DIA 5: <ol><li> En el quinto día después de la siembra realizar un cambio de medio completo para eliminar las células no adherentes. Para ello pre-calentar 20 ml de EGM-2 a 37 ° C en un baño de agua y preparar dos pipetas de 25 ml. </li><li> Retire el medio completo con una pipeta de 25 ml y añadir nuevos, pre-calentado EGM-2 complementado tan pronto como sea posible. Las células no se debe dejar sin medio de más de un minuto para evitar que se seque. Coloque de nuevo en la incubadora y repetir detección del frasco al día. </li><li> Alimentar a las células mediante la sustitución de 30% de medio fresco suplementado por EGM-2 veces por semana. </li></ol> DÍA 14-28: <ol><li> Las colonias típicas con la morfología de adoquines debe aparecer alrededor del día 12. Marque la parte superior externa de la botella para indicar la posición de cada colonia. Permitir a las colonias a crecer hasta el día 28 a menos que las células individuales comienzan a desprenderse. Una vez, las colonias de primaria han sido identificados que pueden ser cosechadas entre los días 14 y 28, dependiendo de su tamaño. </li></ol> B.) ECFC COSECHA <ol><li> Pre-caliente 5 ml de tripsina 0,05% / EDTA, 20 ml y 5 ml de PBS fresco suplementado EGM-2. </li><li> Eliminar por completo medio de la mufla en un tubo Falcon (tenga cuidado de no tocar la superficie de la botella para evitar daños en cualquiera de sus colonias potenciales) y lavar cuidadosamente con células ml de PBS 10 precalentado. </li><li> Añadir 5 ml de tripsina / EDTA y se incuba durante 5 minutos en la incubadora. Las células no deben ser expuestos a la tripsina por más de 7 minutos, ya que se vuelve tóxico. Bien tocando los lados del frasco de ayuda a las células para separar. Apagar la actividad de la proteasa tripsina mediante la adición de aproximadamente 10 ml del medio de cultivo utilizado conservado. </li><li> Levantar la suspensión celular en un Falco 50 mln del tubo. Lavar el matraz una vez con 10 ml de PBS, que es a partir de entonces a la suspensión de células cosechadas. Se centrifuga la suspensión celular a 300 g durante 7 min a 4 ° C para recoger las células y eliminar la tripsina. Eliminar el sobrenadante y resuspender el botón celular de inmediato en 1 ml de PBS. Mantener en hielo. </li><li> Determinar el número de células como se describe en un protocolo JoVe por Ricardo R. y K. Phelani 5. </li></ol> C) ECFC EXPANSIÓN <ol><li> Suplemento de 500 ml EGM-2 como se describe en un 1,1). Pre-caliente a 37 ° C en un baño de agua. </li><li> Por espacio de aproximadamente 2.500 cm 2 preparar una cultura o 11 T220 (225 cm 2) o 15 T175 (175 cm 2) o una de cuatro capas de células de fábrica (CF-4, Nunc, Naperville, IL; <a href="http://www.nuncbrand.com/">http://www.nuncbrand . com</a> ). Además se necesitan dos frescos de ventilación, las tapas y un embudo estéril especial, en el banco de flujo laminar. </li><li> Para ECFCs semilla a una densidad celular inicial de 100 c / cm 2 el cálculo del número de células es necesario. Por un CF-4 252 800 células se resuspenden en 500 ml fresca EGM-2 y se vierte en el CF-4 utilizando el embudo estéril. Cerca de un respiradero de ventilación con la capitalización y la inclinación de la CF-4 a un lado de longitud, lo que permite una distribución equitativa del medio entre los 4 harinas. A continuación, la inclinación de la cámara posterior y abra la tapa del respiradero-cap. Lugar CF-4 en la incubadora. </li><li> 20% de cambio del medio de por lo menos una vez por semana hasta el CF-4 es confluente. </li><li> Las células de la cosecha como se describe en B) mediante el uso de 70 ml de tripsina / EDTA y aproximadamente 200 ml de PBS. </li></ol> Los resultados representativos El uso de este método de aislamiento se observó una formación de colonias de primaria en el día 12. En un estudio anterior fueron capaces de recuperar una media de 4,0 ± 0,8 ECFC-colonias derivadas de cada ml de sangre periférica. ECFCs 4 mostró un aspecto típico de adoquines. Después de la expansión a gran escala, con una densidad de siembra de 100 células / cm 2, se obtuvo&gt; 1 x 10 8 células en un total de aproximadamente 30 días a partir de 3 hombres y 1 mujeres voluntarias (edad entre 26 y 50 años). ECFC cultivadas en estas condiciones, se demostró que proliferativa, funcional y estable genómicamente. 4 Además las células pueden ser almacenadas (en el 10% DMSO) en nitrógeno líquido para su uso posterior 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>

Este aislamiento novela y el método de expansión es un proceso sencillo y eficiente que es más eficiente y menos tiempo, evitando la separación de células (no hay gradiente de densidad es necesario) y menos costoso que las técnicas convencionales. Mediante el uso de contacto pHPL a los antígenos de la especie bovina y xenoimmunization siguientes supuestos se excluye. Durante el período de cultivo pequeños coágulos se pueden formar, causada por la pHPL. Basándonos en nuestra experiencia estos coágulos no afec...

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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>
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		<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

Este documento presenta una estrategia de recuperación de la novela de las células endoteliales progenitoras que forman colonias (ECFCs) de heparina pero por lo demás no manipulado adultos sangre periférica humana dentro de una media de 12 días. Después de la expansión a gran escala&gt; 1x10 8 ECFCs se puede obtener por otras pruebas. Ventajosamente con pHPL el contacto de las células humanas con suero bovino antígenos pueden ser excluidos. Por citometría de flujo e inmunohistoquímica las células aisladas pueden ser caracterizados como ECFC y su funcionalidad in vitro para formar las estructuras vasculares, como puede ser probado en un ensayo de matrigel. Además, estas células pueden ser inyectada por vía subcutánea en un modelo de ratón para la formación de vasos funcionales, perfundidos in vivo. Después de la expansión a largo plazo y la proliferación de criopreservación, la función y la estabilidad genómica parece ser preservado 3,4. Este aislamiento de proteína animal libre y el método de expansión es de fácil aplicación para generar una gran cantidad de 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

Endoteliales que forman colonias células progenitoras (ECFCs) son una herramienta prometedora para estudiar la homeostasis vascular y la reparación.<sup&gt; 1,2</sup&gt; Este artículo presenta un novedoso método de suero de origen animal libres para el aislamiento y la expansión de ECFC de heparina para adultos de sangre periférica humana con agregados de lisado de plaquetas humanas (pHPL) disminuyendo el riesgo de la vacunación anti-bovina.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

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 vistas.  Medical University of Graz, Austria. Endoteliales que forman colonias células progenitoras (ECFCs) son una herramienta prometedora para estudiar la homeostasis vascular y la reparación. 1,2 Este artículo presenta un novedoso método de suero de origen animal libres para el aislamiento y la expansión de ECFC de heparina para adultos de sangre periférica humana con agregados de lisado de plaquetas humanas (pHPL) disminuyendo el riesgo de la vacunación anti-bovina.

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.

El aislamiento de las células endoteliales de vena umbilical (HUVEC)

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

Investigación

Biología

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.

A gran escala de pantallas de bibliotecas metagenómicas

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.

Aislamiento y suero animal expansión libre de cordón umbilical humano derivado de células mesenquimales del estroma (MSC) y endoteliales formadoras de colonias Células Progenitoras (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.

Aislamiento de células endoteliales valvular

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.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

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.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

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 análisis cuantitativo de la insulina que expresan la formación de colonias de progenitores

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.

El aislamiento de células madre CD133 + hígado para la expansión clonal

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.

Caracterización fenotípica y funcional de las células endoteliales Formadoras de Colonias derivados de la sangre del cordón umbilical

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 Aislamiento de Primaria cultivos de fibroblastos humanos

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