Beoordeling van de immuno-modulerende eigenschappen van humane mesenchymale stamcellen (MSC)
Summary
De immuunmodulerende eigenschappen van menselijke mesenchymale stamcellen (MSC) lijken steeds belangrijker voor klinische toepassing. Het gebruik van een co-kweeksysteem van MSCs en perifeer bloed leukocyten vooraf gekleurd met de fluorescerende kleurstof carboxyfluoresceïne succinimidylester (CFSE), beschrijven we de in vitro beoordeling van MSC immunomodulatie bij effector leukocyt proliferatie en specifieke subpopulaties.
Abstract
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.
Introduction
Menselijke mesenchymale stamcellen (MSCs) zijn somatische voorlopers die kunnen differentiëren tot het paraxiale mesodermale lineages bot, kraakbeen en vetweefsel 1-4, alsmede enkele extramesodermal lineages 5. Eerst geïsoleerd uit volwassen beenmerg, zijn deze multilineage progenitors nu gevonden in talrijke weefsels 6-8 en onverwachts aangetoond sterke immunomodulerende eigenschappen die zeer ontvankelijk zijn voor klinische toepassing 12/09 weergegeven zijn. Gedetailleerde mechanismen betrokken bij de immunomodulerende effecten actief onderzocht voor effectieve uitvoering op bepaalde ziektebeelden. Een van de eenvoudigste manieren om immunomodulatie te beoordelen is door beoordeling van de onderdrukking van effector leukocyt proliferatie 13. De meeste effector leukocyten zoals T-lymfocyten en monocyten prolifereren prodigiously wanneer gestimuleerd of geactiveerd. Immunomodulerende functie kan worden beoordeeld bij de onderdrukking vanproliferatie blijkt.
Traditioneel is effector leukocyt proliferatie geëvalueerd door detectie van [3 H] thymidine incorporatie in DNA. Deze methode heeft belangrijke nadelen vanwege de zorgen van bestraling en na gebruik verwijdering, en de complexe apparatuur nodig. Hoewel er niet-radioactieve assays celproliferatie te beoordelen, de succinimidyl ester carboxyfluoresceïne (CFSE) test heeft andere voordelen zoals ter identificatie van specifieke cellulaire populaties, wat vooral nuttig in co-kweek-experimenten waarbij meerdere celtypen. CFSE is een fluorescente kleurstof die cellen kan worden bepaald door flow cytometrische analyse. Als cellen delen, wordt de intensiteit van de cellulaire label proportioneel verminderd; Dit maakt niet alleen de bepaling van de totale celproliferatie maar ook zorgt voor de beoordeling van het aantal celdelingen tot 8 divisies voordat de fluorescentie wordt het moeilijk om detect tegen achtergrond signaal. Bovendien is de stabiliteit van de fluorescerende CFSE maakt in vivo volgen van gemerkte cellen, die cellen kunnen worden gevisualiseerd vele maanden 14.
Deze test kan ook worden gevarieerd om bepaalde soorten effector leukocyten of immunomodulerende functie van specifieke populaties van evaluatie MSC-geïnduceerde immunomodulerende leukocyten-zoals interleukine-10 (IL-10) producerende CD14 + monocyten 15 door het uitvoeren van magnetische korrels oppervlaktemerker winkelwagentje celpopulaties van belang voor of na co-cultuur geschikt. Ons protocol beschrijft de basis assay beoordeling van de immunomodulerende effecten van MSCs voor effector leukocyten (stroomdiagram getoond in figuur 1) en een variatie op deze basis assay voor evaluatie van MSC-geïnduceerde leukocyt immunomodulatie bij allogene CD4 + effector T lymfocyten (getoonde stroomschema in figuur 4).
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).
Materials
| Ficoll-Paque PLUS | GE Healthcare | 71-7167-00 AG | Density grandient for isolation of peripheral blood mononuclear cells (PBMCs) |
| Vibrant CFDA-SE Cell Tracer Kit (CFSE) | Life Technologies | V12883 | Cellular label for detection of cell division |
| Phytoagglutinin (PHA) | Sigma | L8902 | Activation of human PBMCs |
| Dynabeads Human T-Activator CD3/28 | Life Technologies | 111.32D | Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc. |
| autoMACS™ Separator | Miltenyi Biotec | autoMACS™ Separator | Magnetic based cell separator |
| autoMACS® Columns | Miltenyi Biotec | 130-021-101 | separation columns |
| CD14 microbeads, human | Miltenyi Biotec | 130-050-201 | For positive selection of CD14+ human monocytes and macrophages from PBMCs |
| CD4 microbeads, human | Miltenyi Biotec | 130-045-101 | For positive selection of CD4+ human T lymphocytes from PBMCs |
| RPMI 1640 Medium | Life Technologies | 11875 | Human PBMC/leukocyte culture medium |
| DMEM, Low glucose, pyruvate | Life Technologies | 11885 | Human mesenchymal stem cell (MSC) culture medium |
| L-glutamine | Life Technologies | 25030-081 | Supplementation for MSC complete medium |
| Penicillin/Streptomycin | Life Technologies | 15070-063 | Supplementation for PBMC/leukocyte and MSC complete medium |
| Fetal bovine serum (FBS) | 1) Hyclone, for MSC culture 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations) | 1) SH30070.03M 2) 10091-148 | Pre-test lots for support of MSC in vitro culture |
| 24-well cell culture plate | Corning | COR3524 | Co-culture plate |
| 50 mL centrifuge tube | Corning | 430291 | Isolation PBMCs from whole blood by Ficoll-Paque PLUS |
| 15 mL centrifuge tube | Corning | 430766 | Collection of the labeled and unlabeled cell fractions |
| Round-bottom tubes | BD Falcon | 352008 | Collection of cells for flow cytometric analysis |
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