Pre-conditioning mesenchymal stromal cell spheroids for immunomodulatory paracrine factor secretion

Background aims. Mesenchymal stromal cells (MSCs) exhibit the inherent potential to regulate multiple signaling pathways and cell types that contribute to the pathogenesis of inflammatory and immune diseases. However, more recent studies have suggested that the secretion of immunomodulatory factors by MSCs can be enhanced by three-dimensional aggregation or pro-inflammatory cytokine treatment. Methods. Human MSC spheroids were formed by forced aggregation into agarose micro-wells and subsequently cultured in either minimal essential medium alpha supplemented with fetal bovine serum or serum-free, defined MesenCult-XF medium (STEMCELL Technologies, Vancouver, Canada). A subset of the spheroids were treated with pro-inflammatory cytokines interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha or both for 4 days. Immunomodulatory factor (prostaglandin E-2, indoleamine 2,3-dioxygenase, transforming growth factor-beta 1 and interleukin-6) secretion was quantified after 4 days of culture, and the immunomodulatory activity of MSCs was assessed by quantifying activated macrophage expression of TNF-alpha after trans-well co-culture. Results. Culturing human MSCs as three-dimensional aggregates increased secretion of immunomodulatory paracrine factors, which was enhanced further by treatment with IFN-gamma and TNF-alpha, demonstrating that these parameters can synergistically enhance endogenous human MSC immunomodulatory properties. However, immunomodulatory factor secretion was found to be highly dependent on the composition of cell culture medium. Human MSCs cultured in MesenCult-XF medium displayed significantly less expression of prostaglandin E-2, indoleamine 2,3-dioxygenase, transforming growth factor-beta 1 and interleukin-6 compared with human MSCs cultured in medium supplemented with fetal bovine serum. Finally, pre-conditioning of human MSC spheroids with IFN-gamma and TNF-alpha resulted in greater immunomodulatory activity in a macrophage co-culture assay. Conclusions. Altogether, engineering the environment of human MSCs to develop pre-conditioning strategies for enhancing human MSC immunomodulation may be a simple approach for improving MSC-based therapies for the treatment of inflammatory and immune diseases.