Secretome of Aggregated Embryonic Stem Cell-Derived Mesenchymal Stem Cell Modulates the Release of Inflammatory Factors in Lipopolysaccharide-Induced Peripheral Blood Mononuclear Cells

Abstract

Background: Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1β, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs).

Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation.

Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p < 0.0001) and IL-1β (485.2 ± 48.38, p < 0.001) from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome (TNF-α: 166.6 ± 8.04, IL-1β: 125.2 ± 2.73).

Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs.

Keywords: Cell aggregation; Embryonic stem cells; Mesenchymal stem cells; Inflammation; Mononuclear leukocyte.

Fig. 1

Analysis of surface antigens. Flow cytometry of ESC-MSCs was performed with phycoerythrin (PE)-conjugated antibodies for CD73 and CD105 and FITC-conjugated antibodies for CD34 and CD45 markers. The expression of isotype controls is shown as green histograms.

Fig. 2

In vitro differentiation of hESC-MSCs. Osteogenic and adipogenic differentiation are presented by Alizarin red (A and B) and Oil Red O (C and D) staining, respectively. Cells cultured in non-adipogenic and -osteogenic media were considered as controls.

Fig. 3

hESC-MSC aggregation. Cells in adherent (A) and non-adherent culture (B), scale bars = 100 um; (C) schematic representation of the non-adherent culture method; (D) the growth profile of hESC-MSCs in adherent (red) and non-adherent (green) cultures.

Fig. 4

Effects of secretory molecules derived from hESC-MSC aggregates on PBMCs. (A) Schematic representation of the in vitro model to assess anti-inflammatory effects of hESC-MSCs. (B) ELISA assay to study IL-1β, (C) IL-10, and (D) TNF-α secretion from PBMCs after treatment with secretome prepared from non-adherent and adherent culture of hESC-MSC. PBMCs with no treatment were used as control group. Aggregate-CM, LPS-induced PBMCs treated with aggregated MSCs-derived secretome; adherent-CM, LPS-induced PBMCs treated with adherent MSCs-derived secretome; no treatment, untreated LPS-induced PBMCs; Bars are mean ± SD, one-way ANOVA with Turkey’s tests were used for multiple comparisons, n = 3, ^***p < 0.001, ^****p < 0.0001. ns, non-significant