Samantha Khoury et Pamela Ajuyah sont pris en charge par le Postgraduate Award australien. Nous tenons également à remercier le Réseau de recherche translationnelle sur le cancer, Centre de recherche sur le cancer Lowy, l&#39;Université de Nouvelle-Galles du Sud et l&#39;Unité de recherche translationnelle du cancer du Nord pour leur soutien supplémentaire de Samantha Khoury.

Remarque: les échantillons de sérum humain provenant de patients sains ou des patients atteints de cancer ont été obtenus avec le consentement éclairé sous protocoles éthiques humaines agréés du Royal Prince Alfred Hospital de Sydney (numéro de protocole X10-0016 et HREC/10/RPAH/24) et l&#39;Université de Technologie, Sydney. Des échantillons de sérum ont été prélevés chez des patients avant la chirurgie de divers hôpitaux de Sydney et placés dans le stockage à -80 ° C. 1. Petite isolement d&#39;ARN à partir de sérum L&#39;ARN total a été préparé à partir de sérum humain à l&#39;aide d&#39;une version modifiée du protocole du Tri-Reagent LS RT. <ol><li> Décongeler échantillon de sérum congelé sur de la glace, puis transférer 400 pl de sérum fraîchement décongelé dans un tube à centrifuger étiqueté. </li><li> Diluer le sérum avec 100 ul de RNase H 2 O et ajouter la proteinase K à une concentration de 1 mg / ml. </li><li> Incuber à 37 ° C pendant 20 min pour permettre la digestion des protéines par la protéinase K. </li><li> To assurer la solubilisation complète, ajouter 1,5 fois son volume de Tri-Reagent RT LS et 100 pi de 4 bromoanisole. </li><li> Inverser brièvement l&#39;homogénat, effectuer pipetage répétitif pendant 5 secondes et transférer dans un tube de 2 ml lourd à verrouillage de phase marquée. </li><li> Spin l&#39;homogénat à 12 000 g pendant 20 min à 4 ° C. </li><li> Décanter avec soin au moins 1 ml de la solution aqueuse obtenue dans un nouveau tube ADN LoBind. L&#39;interphase organique et doit être pris au piège sous le gel blanc du tube à verrouillage de phase. </li><li> Ajouter 5,0 pl de glycogène (5 mg / ml) et 500 ul d&#39;isopropanol à 100% à la solution aqueuse, mélanger par retournement, et incuber O / N à 20 ° C. </li><li> Suite à O / N incubation, centrifuger l&#39;échantillon pendant 20 min à 12 000 x g dans une centrifugeuse 4 C °. </li><li> Jeter le surnageant clair et effectuer un &quot;flash&quot; rotation pendant 2 min à 16000 x g dans une centrifugeuse C 4 °. </li><li> Retirez délicatement le sol clairution entourant la pastille à l&#39;aide d&#39;une pipette. </li><li> Laver le culot avec 1 ml d&#39;éthanol à 70% et centrifuger à 10 000 g pendant 10 min. Décanter la solution de lavage et répétez l&#39;étape de lavage. </li></ol> 2. ARN remise en suspension dans la solution <ol><li> Reprendre le culot dans 10 ul de RNase H 2 O, en assurant le culot est complètement dissous. Afin de s&#39;assurer que l&#39;ARN est complètement solubilisé l&#39;échantillon peut être chauffé à 55 ° C pendant 5 min. Pendant ce temps, mélanger l&#39;échantillon avec pipetage répété. Pour un rendement élevé de l&#39;ARN total, en commun deux préparations d&#39;ARN provenant du même patient. </li><li> Quantifier l&#39;ARN remises en suspension à l&#39;aide d&#39;un spectrophotomètre UV-Vis et évaluer la qualité de l&#39;ARN en utilisant une Bioanalyseur 2100. Stocker les échantillons d&#39;ARN regroupées à -80 ° C pour une utilisation dans des applications en aval. </li></ol>

<ol>
	<li>Babu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+extraction+efficiency+of+small+RNAs+with+the+Agilent+2100+Bioanalyzer+and+the+Small+RNA+Kit.">Monitoring extraction efficiency of small RNAs with the Agilent 2100 Bioanalyzer and the Small RNA Kit.</a> Agilent Application Note. , (2010).</li><li>Tran, N., Hutvagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+the+regulation+of+the+maturation+of+miRNAs.">Biogenesis and the regulation of the maturation of miRNAs.</a> Essays Biochem. 54, 17-28 (2013).</li><li>Chim, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+placental+microRNAs+in+maternal+plasma.">Detection and characterization of placental microRNAs in maternal plasma.</a> Clin. Chem. 54, 482-490 (2008).</li><li>Lodes, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cancer+with+serum+miRNAs+on+an+oligonucleotide+microarray.">Detection of cancer with serum miRNAs on an oligonucleotide microarray.</a> PLoS One. 4, (2009).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microRNAs+in+serum:+a+novel+class+of+biomarkers+for+diagnosis+of+cancer+and+other+diseases.">Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.</a> Cell Res. 18, 997-1006 (2008).</li><li>Burgos, K. 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Biochem. 162, 156-159 (1987).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+miRNA+processing+and+expression+in+pleomorphic+adenomas+of+the+salivary+gland.">Alterations in miRNA processing and expression in pleomorphic adenomas of the salivary gland.</a> Int. J. Cancer. 124, 2855-2863 (2009).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction:+twenty-something+years+on.">The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.</a> Nat. Protoc. 1, 581-585 (2006).</li><li>Kirschner, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemolysis+during+sample+preparation+alters+microRNA+content+of+plasma.">Haemolysis during sample preparation alters microRNA content of plasma.</a> PLoS One. 6, (2011).</li><li>Murphy, N. R., Hellwig, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+nucleic+acid+organic+extraction+through+use+of+a+unique+gel+barrier+material.">Improved nucleic acid organic extraction through use of a unique gel barrier material.</a> Biotechniques. 21, 934-936 (1996).</li><li>Taylor, C. J., Satoor, S. N., Ranjan, A. K., Pereira e Cotta, M. V., Joglekar, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+measurement+of+noncoding+RNA+in+human+serum.">A protocol for measurement of noncoding RNA in human serum.</a> Experimental Diabetes Research. 2012, (2012).</li><li>Ichikawa, M., Akiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Combination+of+Extraction+Reagent+and+DNA+Microarray+That+Allows+for+the+Detection+of+Global+MiRNA+Profiles+from+Serum/Plasma.">A Combination of Extraction Reagent and DNA Microarray That Allows for the Detection of Global MiRNA Profiles from Serum/Plasma.</a> Methods Mol. Biol. 1024, 247-253 (2013).</li><li>Fromm, B., Harris, P. D., Bachmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+preparations+from+individual+monogenean+Gyrodactylus+salaris-a+comparison+of+six+commercially+available+totalRNA+extraction+kits.">MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits.</a> BMC Res. Notes. 4, 217 (2011).</li><li>Li, Y., Kowdley, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+microRNA+isolation+from+clinical+serum+samples.">Method for microRNA isolation from clinical serum samples.</a> Anal. Biochem. 431, 69-75 (2012).</li><li>McAlexander, M. A., Phillips, M. J., Witwer, K. 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Il n&#39;y a rien à divulguer.

La population de microARN représente environ 0,4-0,5% de l&#39;ARN total dans le sérum. En outre il existe également une teneur élevée en protéine présente dans le sérum humain. Pour améliorer l&#39;ARN et de réduire à la fois la protéine et le phénol contamine nous avons modifié l&#39;approche traditionnelle Chomczynski 9 avec l&#39;ajout de plusieurs étapes. L&#39;ARN total a été isolé à partir de sérum en utilisant la norme Tri-Reagent RT-LS (Centre de reche...

Au cours des dernières années, il ya eu une poussée de plus en plus de découvrir de nouveaux biomarqueurs pour la détection précoce des maladies humaines. Beaucoup d&#39;attention a été porté sur l&#39;utilisation de petits ARN tels que les microARN 2 (miARN ou mirs) comme marqueurs potentiels. Ces petits ARN sont trouvés dans les fluides corporels tels que le sérum, et des études ont montré qu&#39;ils sont résistants à la dégradation et sont stables dans une gamme de conditions environnementales variables 3. Compte tenu de ces caractéristiques miARN, de sérum ou de circulation sont le biomarqueur idéal 4, 5. Actuellement, il existe deux approches principales pour l&#39;isolement de petits ARN à partir de fluides biologiques. La première approche utilise une technologie basée sur les colonnes de lier et éluer les petits ARN 6, tandis que la seconde approche utilise le protocole de longue date avec le phénol et guanidinium thiocyanate réactifs 7. Nous avons développé un protocole simple, efficace, sans colonne pour isoler de petits ARN à partir de sérum humain. L&#39;ARN isolé est immédiatementment utilisable dans des applications en aval, y compris les tableaux ADN d&#39;oligonucléotides et le séquençage de l&#39;ARN. Ce protocole a été développé parce que nous avons été confrontés à plusieurs problèmes lors de l&#39;utilisation des méthodes à base de phénol pour isoler l&#39;ARN à partir de sérum. L&#39;approche traditionnelle Chomczynski est fréquemment utilisé dans la plupart des laboratoires avec une gamme de réactifs disponibles dans la plupart des fournisseurs commerciaux. Toutefois, compte tenu de leur utilisation généralisée, les lignes directrices strictes n&#39;ont pas été développées pour produire toujours ARN de haute qualité à partir de fluides corporels, dans le sang ou le sérum particulier. Les problèmes courants associés à l&#39;isolement d&#39;ARN à partir de sérum d&#39;ARN comprennent des rendements faibles et une contamination par des réactifs utilisés au cours de l&#39;isolement, en particulier le phénol. Notre approche élimine ces impuretés phénoliques pour fournir de l&#39;ARN de haute qualité pour analyse en aval telles que la PCR quantitative (qPCR) et le séquençage de l&#39;ARN. Nous avons également testé cet ARN sur les tableaux de miARN.

<table border="0" cellpadding="0" cellspacing="0" style="width:852px;" width="851">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Name of the Material/Equipment&#160;</td>
			<td style="width:223px;">Company&#160;</td>
			<td style="width:123px;">Catalog Number&#160;</td>
			<td style="width:248px;">Comments/ Description (optional)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Tri-Reagent RT LS</td>
			<td style="width:223px;">Molecular Research Centre, USA</td>
			<td style="width:123px;">TR 118</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:259px;">RNase free H20</td>
			<td style="width:223px;">GIBCO Invitrogen</td>
			<td style="width:123px;">10977-023</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Proteinase K&#160;</td>
			<td style="width:223px;">Finnzymes, Finland</td>
			<td style="width:123px;">EO0491</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Heavy Phaselock Tube</td>
			<td style="width:223px;">5PRIME</td>
			<td>2302830</td>
			<td style="width:248px;">2ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">DNA Lobind tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:123px;">0030 108.078</td>
			<td style="width:248px;">1.5ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Glycogen&#160;</td>
			<td style="width:223px;">Invitrogen, USA</td>
			<td style="width:123px;">10814-010</td>
			<td style="width:248px;">5mg/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Isopropanol&#160;</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">I9516</td>
			<td style="width:248px;">100%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Refrigerated Centrifuge</td>
			<td style="width:223px;">John Morris</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Nanodrop UV-Vis spectrophotometer</td>
			<td style="width:223px;">Thermo Fisher Scientific, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Ethanol</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">E7023</td>
			<td style="width:248px;">70%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Agilent 2100 Bioanalyser</td>
			<td style="width:223px;">Agilent, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
	</tbody>
</table>

isolation of small noncoding rnas from human serum

L&#39;analyse de l&#39;ARN et son expression est une caractéristique commune à de nombreux laboratoires. D&#39;importance est l&#39;émergence de petits ARN comme les microARN, qui se trouvent dans les cellules de mammifères. Ces petits ARN sont des régulateurs de gènes puissants de contrôle des canaux vitaux tels que la croissance, le développement et la mort et beaucoup d&#39;intérêt a été réalisé à leur expression dans les fluides corporels. Ceci est dû à leur dérégulation dans les maladies humaines telles que le cancer et leur application possible en tant que biomarqueurs sériques. Cependant, l&#39;analyse de l&#39;expression des miARN dans le sérum peut être problématique. Dans la plupart des cas, la quantité de sérum est de limiter et de sérum contient de faibles quantités d&#39;ARN total, dont de petits ARN constituent seulement 0,4-0,5% 1. Ainsi l&#39;isolement de quantités suffisantes d&#39;ARN de qualité à partir de sérum est un défi majeur pour les chercheurs d&#39;aujourd&#39;hui. Dans ce document technique, nous démontrons une méthode qui utilise seulement 400 pi de sérum humain pour obtenir l&#39;ARN est suffisante pour soit des puces à ADN ou analyse qPCR. L&#39;unvantages de ce procédé sont sa simplicité et sa capacité à produire de l&#39;ARN de haute qualité. Il ne nécessite pas de colonnes spécialisées pour la purification de petits ARN et utilise des réactifs généraux et du matériel trouvés dans des laboratoires communs. Notre procédé utilise un gel de verrouillage de phase pour éliminer la contamination de phénol tout en donnant en même temps l&#39;ARN de haute qualité. Nous introduisons également une étape supplémentaire pour éliminer encore tous les contaminants lors de l&#39;étape d&#39;isolement. Ce protocole est très efficace pour isoler l&#39;ARN total des rendements allant jusqu&#39;à 100 ng / ul de sérum, mais peut également être adapté à d&#39;autres tissus biologiques.

La figure 1 représente un spectre typique UV / Vis de l&#39;ARN isolé à partir de sérum. De ce profil, nous avons constaté la contamination de la protéine à 280 nm avec du phénol et de contaminants organiques à la fois à 270 nm et 230 nm, respectivement. Résidu thiocyanate de guanidinium a également été noté à 260 nm. Afin de réduire les contaminants, une série d&#39;étapes d&#39;optimisation ont été apportées à la procédure de Tri-Reagent RT-LS standard. On a ajouté 5 mg / ml d...

Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

Isolation of Small Noncoding RNAs from Human Serum

Ce protocole décrit un procédé d&#39;extraction de petits ARN à partir de sérum humain. Nous avons utilisé cette méthode pour isoler les micro-ARN à partir de sérum de cancer destiné à être utilisé dans les puces à ADN et PCR quantitative singleplex également. Le protocole utilise le phénol et du thiocyanate de guanidinium réactifs avec des modifications pour donner l&#39;ARN de haute qualité.

Isolation des petits ARN non codant de sérum humain

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

isolation-of-small-noncoding-rnas-from-human-serum

Research

JoVE Journal

Biology

18.0K vues.  University of Technology, Sydney.  Ce protocole décrit un procédé d'extraction de petits ARN à partir de sérum humain. Nous avons utilisé cette méthode pour isoler les micro-ARN à partir de sérum de cancer destiné à être utilisé dans les puces à ADN et PCR quantitative singleplex également. Le protocole utilise le phénol et du thiocyanate de guanidinium réactifs avec des modifications pour donner l'ARN de haute qualité. 

Vidéo: Isolation of Small Noncoding RNAs from Human Serum

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Isolement des noyaux neuronaux à partir de tissus post-mortem du cerveau de l&#39;homme

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Recherche

Biologie

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolement des cellules souches de l&#39;homme xénogreffes de cancer du pancréas

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolation Manuel des cellules souches du tissu adipeux de Lipoaspirates humaines

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Isolation des myofibroblastes primaires de souris et de tissus du côlon humain

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Isolement de myofibroblastes de Mouse et œsophage humain

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolement des cardiomyocytes ventriculaires humains des tranches myocardales vibratoires-cut

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

Isolement des neutrophiles humains du sang total et des buffys

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Isolement des cellules épithéliales du follicule dentaire humain

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...