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| MSC Suppression Inspector, human |
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| Overview |
| The MSC Suppression Inspector was developed for the functional characterization of human mesenchymal stem cells, also known as mesenchymal stromal cells (MSCs) by in vitro suppression assays. |
| Details |
Background information
MSCs are fibroblast-like plastic-adherent cells that can be isolated from a variety of tissues, such as bone marrow or adipose tissue. During the last few years the attention of scientists was redirected away from the multipotentiality of MSCs towards their possibility for immunomodulation. It was observed that bone marrow derived MSCs suppress T-cell proliferation1,2. This function of MSCs can be analyzed using the MSC Suppression Inspector which contains an optimal T cell stimulation reagent for an MSC suppression assay.
Detailed procedure
The MSC Suppression Inspector consists of Anti-Biotin MACSiBead Particles that are pre-loaded with biotinylated CD2, CD3, and CD28 antibodies. MSCs are co-cultured with CD4+CD25– or CD4+ responder T cells (Tresp) at different ratios in the presence of a polyclonal stimulus, in this case the MSC Suppression Inspector. Tresp cells alone show a proliferative response. Co-culture of MSCs with Tresp cells results in reduced proliferation of Tresp cells. Cell proliferation is determined by 3H-thymidine incorporation or detected by carboxyfluorescein succinimidyl ester (CFSE) staining. |
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| Figure 1 |
MSCs were isolated from human bone marrow and expanded with MACS NH Expansion Medium. After two passages MSCs were co-cultured with CD4+CD25– responder T cells (Tresp) at different ratios. For T cell stimulation, the MSC Suppression Inspector was added to the culture. Proliferation of T cells was determined by 3H-thymidine incorporation.
Tresp show high proliferation after stimulation with the MSC Suppression Inspector (A). When adding MSCs, Tresp proliferation is suppressed dramatically (B). Unstimulated Tresp show no proliferation (D). MSCs alone show little proliferation with or without stimulation (C and E). |
| Tritium-based in vitro suppression assay |
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| Figure 2 |
MSCs were isolated from human bone marrow either by plastic adherence (PA-MSCs) or by isolation (CD271+ MSCs) using the CD271 MicroBead Kit (APC). Both PA-MSCs and CD271+ MSC were expanded with MACS NH Expansion Medium. After two passages MSCs were co-cultured with CFSE-labeled CD4+CD25– responder T cells (Tresp). For T cell stimulation, the MSC Suppression Inspector was added to the cultures. The percentage of proliferating Tresp was measured as CFSE dye dilution analyzed by flow cytometry using the MACSQuant® Analyzer.
Almost 90% of all Tresp proliferate after stimulation with the MSC Suppression Inspector (A). When adding PA-MSCs, Tresp proliferation is suppressed to a level of about 28% (B). The addition of CD271+ MSCs suppresses the Tresp proliferation to a level of 20% (D). MSCs alone show no proliferation with or without stimulation (C, E, G, and H). Unstimulated Tresp show a proliferation rate of 30% (F). |
| CFSE-based in vitro suppression assay |
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| Details |
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| References |
| 1. Di Nicola, M. et al. (2002) Blood 99: 3838–3843. |
| 2. Bartholomew, A. et al. (2002) Exp. Hematol. 30: 42–48. |
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