Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming

Abstract

BackgroundMesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures.MethodsWe established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures.Results3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6.ConclusionsIncreased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

“…We investigated the secretion of cytokines such as VEGF and IL-6 from MSC spheroids, as senescence in monolayer culture is often characterized by measuring the SASP. 25 However, the secretome of MSC spheroids differs significantly from cells in monolayer, 26,27 limiting our ability to use changes in the secretome to definitively describe senescence. We observed decreases in VEGF ( Figure 2A other reports identifying TNTs.…”

Mircrining the injured heart with stem cell-derived exosomes: an emerging strategy of cell-free therapy

“…Priming 3Dcultured human MSCs with interleukin-1 enhanced the paracrine release of GCSF, VEGF, and interleukin-1 receptor antagonist (IL-1Ra). Protein array showed a more potent immune profile which was required to orchestrate an effective tissue repair [27]. The effect of culture surface topology and microenvironment significantly alters the morphology of the cultured cells besides altering their cellular activity including the secretome profile [28].…”

Three-Dimensional Culture of Umbilical Cord Mesenchymal Stem Cells Effectively Promotes Platelet Recovery in Immune Thrombocytopenia

“…In addition, polymer scaffold and perfusion-based 3D culture systems are used. 35,[50][51][52][53] MSCs can maintain the structural basis of cell-cell and cell-matrix contact by aggregation, thereby preventing cell loss due to apoptosis and improving implantation into host tissues. 54) Our experimental results show that MSCs in the 3D group express higher levels of ECM factors (e.g., CoL-I, fibronectin, and integrin), and more effectively resist IFN-γ-induced apoptosis.…”