Os autores agradecem a Dra. Katharina Schallmoser para fornecer pHPL e Margaretha Frühwirth e Daniela Thaler para assistência técnica excelente.

A.) ECFC ISOLATON DIA 1: <ol><li> Antes de começar a preparar com a experiência da célula endotelial Crescimento meio de cultura Médio-2 (EGM-2). Suplemento 500ml endotelial Basal Medium-2 com heparina (1ml de 1000 U / ml), solução de 100x 5ml de penicilina / estreptomicina (todos Sigma, St. Louis, MO; <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamina (concentração final de 2 mM ), e 50 ml de lisado de plaquetas agrupadas humano (pHPL). Em seguida, adicionar 0,2 ml de hidrocortisona, 2 ml hFGF-B, 0,5 ml VEGF, EGF 0,5 ml, 0,5 ml de IGF-1 e 0,5 ml de ácido ascórbico (fornecido como SingleQuots, todos Lonza, Walkersville, MD; <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience. com /</a> ). Estéril filtrado o meio através de um m-pore size 0,2 500 ml de filtro a vácuo Millipore. Desde pHPL formas pequenos coágulos que podem precisar de dois filtros para um médio. Pré-aquecer filtrada EGM-2 para 37 ° C em banho-maria </li><li> Coletar as amostras de sangue, chamando 5 ml de sangue periférico humano adulto a partir da veia cubital em um tubo de 6ml conservante livre vacutainer heparina sódica-sangue. Você deve processar as amostras de sangue dentro de um máximo de duas horas. </li><li> Comece por preparar um centímetro 75 2 (T75; Costar) frasco com uma tampa de ventilação, uma pipeta 25 ml e dois de 5 pipetas ml em uma bancada de fluxo laminar. </li><li> Pré-preencher 15 ml pré-aquecido complementada EGM-2 para o balão com a pipeta de 25 ml. Agora, abra o frasco da amostra de sangue e todos os 5 ml com a pipeta de 5 ml para o balão. Lavar o frasco vazio de sangue com 5 ml de adicionais EGM-2 com o segundo pipeta de 5 ml. O volume total dentro da T75 é de 25 ml. Feche a tampa de ventilação do frasco e coloque em uma incubadora a 37 ° C, 5% CO 2 e umidade de 95% para 24 horas. </li></ol> 2 º DIA: <ol><li> Após aproximadamente 24 horas (durante a noite), você deve remover a mistura meio-sangue para se livrar de células não aderentes. Para este preparar duas pipetas de 25 ml e três pipetas 5 ml no fluxo laminar, pré-aquecer 20 ml de suplementada EGM-2 (preparada no dia anterior) e 30 ml de PBS a 37 ° C em banho-maria. </li><li> Retirar o balão com a amostra da incubadora e remover todos os sobrenadante com uma pipeta de 25 ml. Tome cuidado para não arranhar a superfície frasco para evitar danificar qualquer colônia potencial. Lavar o frasco de superfície interna adesão celular três vezes suavemente adicionando 10 ml de PBS pré-aquecido e inclinando o frasco de um lado para o outro. Remover o PBS imediatamente cada vez e descarte. </li><li> Adicionar 20 ml de EGM-2 pré-aquecido fresco para o frasco e colocá-lo de volta para a incubadora. Este processo deve ser realizado o mais rápido e suave quanto possível para as células anexado não para separar. ECFCs desprendem facilmente, se forem mantidos em PBS ou à temperatura ambiente para a longa. </li><li> Tela através do frasco sistemicamente dias a cada segundo a partir de dois dias usando uma baixa ampliação de um microscópio de luz procurando conseqüência ECFC. Quando uma colônia é encontrado marca no topo do lado externo do frasco, indicando a posição da colônia. Note que o flaks não deveria estar fora da incubadora por mais de 5 minutos. </li></ol> DIA 5: <ol><li> No quinto dia após a semeadura realizar uma mudança meio completo para remover as células não aderentes. Para este efeito, pré-aquecer 20 ml da EGM-2 para 37 ° C em banho-maria e preparar duas pipetas de 25 ml. </li><li> Remova o meio completo, utilizando uma pipeta de 25 ml e adicionar fresco, EGM-2 pré-aquecido complementado o mais rapidamente possível. Células não deve ser deixado sem meio de mais de um minuto para evitar a secagem. Coloque de volta para a incubadora e repita o frasco de triagem diária. </li><li> Alimentar as células, substituindo 30% da média complementado com novas EGM-2 duas vezes por semana. </li></ol> DIA 14-28: <ol><li> Colônias com morfologia típica de calçada deve aparecer por volta do dia 12. Marcar a parte superior externa do frasco para indicar a posição de cada colônia. Permitir que as colônias de crescer até o dia 28, a menos que uma única célula começa a destacar. Uma vez, colônias primário foram identificados, podem ser colhidas entre os dias 14 e 28, dependendo do seu tamanho. </li></ol> B.) ECFC HARVEST <ol><li> Pré-aquecer 5 ml de tripsina 0,05% / EDTA, 20 ml e 5 ml PBS fresco suplementado EGM-2. </li><li> Remover completamente o meio do balão para um tubo Falcon (tomar cuidado para não tocar na superfície do frasco para evitar danificar qualquer colônia potencial) e lavar cuidadosamente as células com 10 ml PBS pré-aquecido. </li><li> Adicionar 5 ml de tripsina / EDTA e incubar por 5 minutos na incubadora. As células não devem ser expostas à tripsina por mais de 7 min, uma vez que se torna tóxica. Exaustivamente batendo nos lados do frasco ajuda as células a separar. Saciar a atividade de protease tripsina, adicionando cerca de 10 ml do meio de cultura preservada utilizada. </li><li> Anular a suspensão de células em um 50 ml Falcotubo n. Lavar o frasco uma vez com 10 ml PBS que é então adicionado à suspensão de células colhidas. Centrifugar a suspensão celular a 300 g por 7 min a 4 ° C para coletar as células e remover a tripsina. Desprezar o sobrenadante e ressuspender o pellet celular imediatamente em 1 ml de PBS. Manter no gelo. </li><li> Determine o número de células, como descrito em um protocolo JOVE por R. Ricardo e Phelani K. 5. </li></ol> C.) ECFC EXPANSÃO <ol><li> Suplemento 500 ml EGM-2, conforme descrito em A 1.1). Pré-aquecer a 37 ° C em banho-maria. </li><li> Para cerca de 2500 cm 2 de espaço de cultura, quer preparar uma 11 T220 (225 cm 2) ou 15 T175 (175 cm 2) ou uma fábrica de células de quatro camadas (CF-4; Nunc, Naperville, IL; <a href="http://www.nuncbrand.com/">http://www.nuncbrand . com</a> ). Além disso, você vai precisar de dois frescos de ventilação-caps e um funil especial estéril na bancada de fluxo laminar. </li><li> Para ECFCs semente em uma densidade celular inicial de 100 c / cm 2 cálculo do número de células é necessário. Para uma CF-4 252.800 células são ressuspendidas em 500 ml fresco EGM-2 e derramado no CF-4 usando o funil estéril. Fechar um painel com a abertura cap-and-tilt a CF-4 lado longitude para um, permitindo uma distribuição igual do meio entre todas as quatro farinhas. Em seguida, incline a câmara para trás e abrir a tampa do respiradouro-cap. Coloque CF-4, em incubadora. </li><li> Mudança de 20% do meio de pelo menos uma vez por semana até CF-4 é confluente. </li><li> Células colheita, conforme descrito no B.) usando 70 ml de tripsina / EDTA e cerca de 200 ml PBS. </li></ol> RESULTADOS REPRESENTANTE Usando este método de isolamento observou-se uma colônia de formação primária no dia 12. Em um estudo anterior foram capazes de recuperar uma média de 4,0 ± 0,8 ECFC-colônias derivadas de cada ml de sangue periférico. ECFCs 4 mostrou uma aparência de pedra de calçada típica. Após a expansão em grande escala, com uma densidade de semeadura de 100 células / cm 2, obtivemos&gt; 1 x 10 8 células em um total de aproximadamente 30 dias a partir de três voluntários do sexo masculino e 1 feminino (idade entre 26 e 50 anos). ECFC cultivadas sob estas condições se mostraram proliferativa, funcional e genomicamente estável. 4 Além disso as células podem ser armazenadas (em 10% DMSO) em nitrogênio líquido para posterior utilização 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 isolamento romance e método de expansão é um processo simples e eficiente que é mais eficiente e menos morosa, evitando qualquer separação de células (sem gradiente de densidade necessário) e menos caro do que as técnicas convencionais. Por meio de contato pHPL aos antígenos bovina e xenoimmunization subseqüentes putativa é excluído. Durante o período de cultivo pequenos coágulos podem se formar, causada pela pHPL. Baseado em nossa experiência esses coágulos não afetam a formação de colônias e ...

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		<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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<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 trabalho apresenta uma estratégia de recuperação da novela para colônia de células progenitoras endoteliais formando (ECFCs) de heparinizado mas de outra maneira não-manipulado sangue periférico humano adulto dentro de uma média de 12 dias. Após a expansão em grande escala&gt; 1x10 8 ECFCs pode ser obtido para mais testes. Vantajosamente usando pHPL o contato de células humanas com antígenos soro bovino pode ser excluída. Por citometria de fluxo e imunohistoquímica das células isoladas podem ser caracterizadas como ECFC e sua funcionalidade vitro para formar estruturas vasculares, como pode ser testado em um ensaio de Matrigel. Além disso essas células podem ser subcutânea em um modelo de mouse para forma funcional, vasos perfundidos in vivo. Após a expansão a longo prazo e proliferação de criopreservação, função e estabilidade genômica parecem ser preservada 3,4. Este isolamento de proteína animal-livre e método de expansão é de fácil aplicação para gerar uma grande quantidade 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

Endotelial colônia formando células progenitoras (ECFCs) são uma ferramenta promissora para o estudo da homeostase vascular e reparação.<sup&gt; 1,2</sup&gt; Este artigo introduz um novo método de soro animal-livre para isolamento e expansão de ECFC heparinizado de sangue humano adulto periférica com pool de lisado de plaquetas humanas (pHPL) diminuindo o risco de anti-bovino imunização.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

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 visualizações.  Medical University of Graz, Austria. Endotelial colônia formando células progenitoras (ECFCs) são uma ferramenta promissora para o estudo da homeostase vascular e reparação. 1,2 Este artigo introduz um novo método de soro animal-livre para isolamento e expansão de ECFC heparinizado de sangue humano adulto periférica com pool de lisado de plaquetas humanas (pHPL) diminuindo o risco de anti-bovino imunização.

Vídeo: 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 de células endoteliais da veia umbilical humana (HUVEC)

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

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.

Grande Escala Telas de Bibliotecas metagenomic

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 animais de Expansão de soro livre de cordão umbilical humano Derivado células mesenquimais estromais (MSCs) e endoteliais Células Progenitoras Colony Forming (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 de Valvular Células Endoteliais

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 Cultura da Avian embrionárias Células Progenitoras 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.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele 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.

Uma análise quantitativa para expressar Insulin-formadoras de colônias 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.

Isolamento de CD133 + células tronco do fígado de Expansão 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.

Caracterização fenotípica e funcional das células endoteliais Formadoras de Colônias derivado do sangue do cordão umbilical humano

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 de culturas primárias 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 ...