Name: assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)
Uploaded: 2015-12-24T14:00:00
Description: Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

Le consentement éclairé du patient, tel qu&#39;approuvé par le conseil d&#39;examen institutionnel doit être obtenue pour l&#39;utilisation de cellules humaines. 1. Densité isolement Gradient de cellules mononucléaires du sang périphérique humain (CMSP) <ol><li> Ajouter 25 ml du sang total hépariné dans un tube de 50 ml par 25 ml d&#39;une pipette. <ol><li> Diluer les cellules avec 25 ml de solution saline tamponnée au phosphate (PBS). </li></ol></li><li> Ajouter 15 ml d&#39;un ...

<ol>
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Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

Les cellules souches mésenchymateuses humaines (MSC) sont somatiques progéniteurs qui peuvent se différencier en lignées mésodermiques paraxiaux de l&#39;os, le cartilage, le tissu adipeux et 4/1, ainsi que de quelques lignées extramesodermal 5. Première isolé de la moelle osseuse adulte, ces progéniteurs multilignée ont été trouvés dans de nombreux tissus 6-8 et, de façon inattendue, montré pour avoir des propriétés immunomodulatrices fortes qui apparaissent prête très bien à l&#39;application clinique 9-12. Mécanismes détaillés impliqués dans les effets immunomodulateurs sont activement à l&#39;étude pour une application efficace des entités pathologiques spécifiques. Une des façons les plus simples pour évaluer l&#39;immunomodulation est en évaluant pour la suppression de la prolifération des leucocytes effecteur 13. La plupart des leucocytes effectrices telles que les monocytes et les lymphocytes T prolifèrent prodigieusement lorsqu&#39;elles sont stimulées ou activées. Fonction immunomodulatrice peut être évaluée lorsque la suppression deprolifération est mis en évidence. Traditionnellement, les leucocytes effecteur prolifération a été évaluée par détection de la [3H] thymidine dans l&#39;ADN. Cependant, cette méthode présente des inconvénients importants en raison des préoccupations de rayonnement et de l&#39;utilisation post-élimination, ainsi que l&#39;équipement complexe nécessaire. Bien qu&#39;il existe des dosages non radioactifs pour évaluer la prolifération cellulaire, l&#39;ester carboxyfluorescéine succinimidyl (CFSE) dosage présente d&#39;autres avantages tels que permettant l&#39;identification de populations cellulaires spécifiques, ce qui est particulièrement utile dans des expériences de co-culture impliquant plusieurs types de cellules. CFSE est un colorant fluorescent cellulaire qui peut être évaluée par analyse de cytométrie en flux. Comme les cellules se divisent, l&#39;intensité de cette étiquette cellulaire est diminuée proportionnellement; cette détermination permet non seulement de la prolifération cellulaire en général, mais aussi permet d&#39;évaluer le nombre de divisions cellulaires jusqu&#39;à 8 divisions avant la fluorescence devient difficile de detect contre signal de fond. En outre, la stabilité du CFSE fluorescent permet un suivi in vivo de cellules marquées telles que les cellules peuvent être trouvées à de nombreux mois 14. Ce test peut également être modifiée pour évaluer des types spécifiques de leucocytes effectrices ou la fonction immunomodulatrice de populations spécifiques de MSC induite par les leucocytes, tels immunomodulateurs comme l&#39;interleukine-10 (IL-10) la production de monocytes CD14 + 15 en effectuant magnétique bourrelet sélection de marqueur de surface de populations de cellules d&#39;intérêt avant ou après la co-culture selon le cas. Notre protocole décrit l&#39;essai de base de l&#39;évaluation des effets immunomodulateurs de MSC sur les leucocytes effectrices (organigramme représenté sur la figure 1) et une variation sur ce test de base pour l&#39;évaluation du MSC-induite immunomodulation de leucocytes sur CD4 + lymphocytes T effecteurs allogéniques (organigramme représenté la figure 4).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Ficoll-Paque PLUS</td>
			<td style="width:259px;">GE Healthcare</td>
			<td style="width:121px;">71-7167-00 AG</td>
			<td style="width:549px;">Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">V12883</td>
			<td>Cellular label for detection of cell division</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Phytoagglutinin (PHA)</td>
			<td style="width:259px;">Sigma</td>
			<td>L8902</td>
			<td>Activation of human PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Dynabeads Human T-Activator CD3/28</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">111.32D</td>
			<td style="width:549px;">Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">autoMACS&#8482; Separator</td>
			<td>Magnetic based cell separator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">autoMACS&#174; Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td>separation columns</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:276px;">CD14 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td style="width:549px;">For positive selection of CD14+ human monocytes and macrophages from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">130-045-101</td>
			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">RPMI 1640 Medium</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11875</td>
			<td>Human PBMC/leukocyte culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">DMEM, Low glucose, pyruvate</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11885</td>
			<td style="width:549px;">Human mesenchymal stem cell (MSC) culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">L-glutamine</td>
			<td style="width:259px;">Life Technologies</td>
			<td>25030-081</td>
			<td>Supplementation for MSC complete medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/Streptomycin</td>
			<td style="width:259px;">Life Technologies</td>
			<td>15070-063</td>
			<td>Supplementation for PBMC/leukocyte and MSC complete medium</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:276px;">Fetal bovine serum (FBS)</td>
			<td style="width:259px;">1) Hyclone, for MSC culture&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td>
			<td style="width:121px;">1) SH30070.03M 2) 10091-148</td>
			<td>Pre-test lots for support of MSC in vitro culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">24-well cell culture plate</td>
			<td style="width:259px;">Corning</td>
			<td>COR3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">50 mL centrifuge tube</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430291</td>
			<td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">15 mL centrifuge tube&#160;</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430766</td>
			<td>Collection of the labeled and unlabeled cell fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round-bottom tubes</td>
			<td style="width:259px;">BD Falcon&#160;</td>
			<td style="width:121px;">352008</td>
			<td>Collection of cells for flow cytometric analysis</td>
		</tr>
	</tbody>
</table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

La figure 1 représente le schéma d&#39;ensemble de l&#39;expérience, et la figure 2 illustre l&#39;aspect des différentes conditions de culture cellulaire en tant que visualisées par contraste de phase inversé microscopie. MSC sont des cellules adhérentes avec un fibroblastique, la morphologie en forme de fuseau, tandis que les PBMC et les leucocytes sont de petites cellules non adhérentes rondes. Ces deux types de cellules morphologiquement différents peuvent être clairement vus dans la co-cultur...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Les propriétés immunomodulatrices des cellules souches mésenchymateuses humaines (MSC) apparaissent de plus en plus pertinentes pour l&#39;application clinique. L&#39;utilisation d&#39;un système de co-culture de cellules souches mésenchymateuses et leucocytes du sang périphérique pré-colorées avec le succinimidyl ester de carboxyfluorescéine fluorescent du colorant (CFSE), nous décrivons l&#39;évaluation de l&#39;immunomodulation MSC in vitro sur la prolifération des leucocytes et effectrice des sous-populations spécifiques.

Évaluation de la propriétés immunomodulatrices des mésenchymateuses humaines cellules souches (MSC)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

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