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. 2009 Dec;37(12):1445-53.
doi: 10.1016/j.exphem.2009.09.004. Epub 2009 Sep 20.

Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages

Jaehyup Kim  1 , Peiman Hematti
Affiliations

Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages

Jaehyup Kim et al. Exp Hematol. 2009 Dec.

Abstract

Objective: Mesenchymal stem cells (MSCs) are capable of modulating the immune system through interaction with a wide range of immune cells. This study investigates the hypothesis that interaction of MSCs with macrophages could play a significant role in their antiinflammatory/immune modulatory effects.

Materials and methods: MSCs were derived from bone marrow and monocytes were isolated from peripheral blood of healthy donors. We cultured human monocytes for 7 days without any added cytokines to generate macrophages, and then cocultured them for 3 more days with culture-expanded MSCs. We used cell surface antigen expression and intracellular cytokine expression patterns to study the immunophenotype of macrophages at the end of this coculture period, and phagocytic assays to investigate their functional activity in vitro.

Results: Macrophages cocultured with MSCs consistently showed high-level expression of CD206, a marker of alternatively activated macrophages. Furthermore, these macrophages expressed high levels of interleukin (IL)-10 and low levels of IL-12, as determined by intracellular staining, typical of alternatively activated macrophages. However, macrophages cocultured with MSCs also expressed high levels of IL-6 and low levels of tumor necrosis factor-alpha (TNF-alpha) compared to controls. Functionally, macrophages cocultured with MSCs showed a higher level of phagocytic activity.

Conclusions: We describe a novel type of human macrophage generated in vitro after coculture with MSCs that assumes an immunophenotype defined as IL-10-high, IL-12-low, IL-6-high, and TNF-alpha-low secreting cells. These MSC-educated macrophages may be a unique and novel type of alternatively activated macrophage with a potentially significant role in tissue repair.

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Figures

Figure-1
Figure-1
Comparison of expression of CD206 cell surface marker on macrophages co-cultured in direct contact to MSCs (MSC) or within a transwell system (Transwell), and without any MSCs (Control). Results are shown comparing mean fluorescence intensity of CD206 expression.
Figure-2
Figure-2
Gating strategy to separate macrophage from MSC and comparison of expression of IL-10 in MSC co-cultured macrophage versus control macrophage. A) Gating of MSC co-cultured macrophages using CD14 APC and CD90 PerCP-Cy5.5; B) Isotype control to show specificity of IL-10 staining of MSC co-cultured macrophage; C) Expression of IL10 in MSC co-cultured macrophages; D) Expression of IL-10 in control macrophages.
Figure-3
Figure-3
Intracellular cytokine staining of MSC co-cultured and control macrophages. A) MSC co-cultured macrophages stained with anti-IL-12 PE; B) Control macrophage stained with anti-IL12 PE; C) MSC co-cultured macrophage stained with anti-IL6 PE; D) Control macrophage stained with anti-IL6 PE; E) MSC co-cultured macrophage stained with anti-TNF-α PE; F) Control macrophage stained with anti-TNF-α PE.
Figure-4
Figure-4
Fold changes of percentages of MSC co-cultured macrophages versus control macrophage positively stained for the intracellular target cytokine.
Figure-5
Figure-5
Phagocytic assay: A) Gating strategy for MSC co-cultured macrophages looking at CD14+ CD90− cells; B) Gating of control macrophages; C) MSC co-cultured macrophage in gray and control macrophage in white, Incubated at 4° C for 1 hour; D) MSC co-cultured macrophages in gray and control macrophage in white incubated at 37° C for 1 hour.
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