Vurdering af immunmodulerende egenskaber af humane mesenchymale stamceller (MSC'er)
Summary
De immunmodulerende egenskaber af humane mesenkymale stamceller (MSC) fremgår mere relevante for klinisk anvendelse. Ved hjælp af en co-dyrkningssystemet ifølge MSC'er og perifere blodleukocytter præ-farvet med det fluorescerende farvestof carboxyfluorescein succinimidyl ester (CFSE), beskriver vi in vitro vurdering af MSC immunmodulation på effektor leukocyt proliferation og specifikke subpopulationer.
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
Humane mesenchymale stamceller (MSC'er) er somatiske stamceller, der kan differentiere til de akseparallelle mesodermale slægter af knogler, brusk og fedtvæv 1-4, samt et par extramesodermal slægter 5. Først isoleret fra den voksne knoglemarven, har disse multilineage progenitorer nu blevet fundet i mange væv 6-8 og uventet, vist sig at have stærke immunmodulerende egenskaber, der vises meget modtagelig for klinisk anvendelse 9-12. Detaljerede mekanismer involveret i de immunmodulerende effekter er aktivt at blive undersøgt for en effektiv anvendelse af specifikke sygdomstilstande enheder. En af de mest enkle måder at evaluere immunmodulation er ved at vurdere til undertrykkelse af effektor leukocyt spredning 13. De fleste effektor leukocytter såsom T-lymfocytter og monocytter formere uhyre når stimuleret eller aktiveret. Immunmodulerende funktion kan vurderes, når undertrykkelse afproliferation fremgår.
Traditionelt har effektor leukocyt proliferation blevet evalueret ved påvisning af [3H] thymidin-inkorporering i DNA. Men denne metode har betydelige ulemper på grund af de bekymringer af stråling og post-brug bortskaffelse samt det komplekse nødvendige udstyr. Mens der er ikke-radioaktive assays for at vurdere celleproliferation, at carboxyfluorescein succinimidyl ester (CFSE) assay har andre fordele såsom at muliggøre identifikation af specifikke cellulære populationer, hvilket er særligt nyttigt i co-kultur eksperimenter involverer flere celletyper. CFSE er et fluorescerende farvestof, som cellulært kan vurderes ved flowcytometrisk analyse. Som cellerne deler, er intensiteten af denne cellulære label faldet proportionalt; dette giver ikke kun bestemmelse af den samlede celleproliferation, men også giver mulighed for vurdering af antallet af celledelinger op til 8 divisioner, før fluorescensen bliver vanskeligt at Detect mod baggrundssignal. Desuden stabiliteten af det fluorescerende CFSE muliggør in vivo sporing af mærkede celler, således at celler kan visualiseres op til mange måneder 14.
Dette assay kan også varieres for at vurdere specifikke typer af effektorceller leukocytter eller immunmodulerende funktion af specifikke populationer af MSC-induceret immunmodulerende leukocytter-såsom interleukin-10 (IL-10) produktion af CD14 + monocytter 15 ved at udføre magnetisk perle overflademarkør udvælgelse af cellepopulationer af interesse før eller efter co-kultur som passende. Vores protokol beskriver den grundlæggende analyse af vurderingen af de immunmodulerende virkninger af MSC'er på effektor leukocytter (rutediagram vist på figur 1) og en variation af denne grundlæggende assay til evaluering af MSC-induceret leukocyt immunmodulation på allogene CD4 + T-lymfocytter (rutediagram vist i figur 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…
Disclosures
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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